Discovery of N-[4-[6-tert-butyl-5-methoxy-8-(6-methoxy-2-oxo-1 H -pyridin-3-yl)-3-quinolyl]phenyl]methanesulfonamide (RG7109), a potent inhibitor of the hepatitis C virus NS5B polymerase

Article

pubs.acs.org/jmc

Discovery of N‑[4-[6-tert-Butyl-5-methoxy-8-(6-methoxy-2-

oxo‑1H‑pyridin-3-yl)-3-quinolyl]phenyl]methanesulfonamide

(RG7109), a Potent Inhibitor of the Hepatitis C Virus NS5B

Polymerase

Francisco X. Talamas,* Sarah C. Abbot, Shalini Anand, Ken A. Brameld, David S. Carter, Jun Chen,

Dana Davis, Javier de Vicente, Amy D. Fung, Leyi Gong, Seth F. Harris, Petra Inbar, Sharada S. Labadie,

Eun K. Lee, Remy Lemoine, Sophie Le Pogam, Vincent Leveque, Jim Li, Joel McIntosh, Isabel Najera,

́

Jaehyeon Park, Aruna Railkar, Sonal Rajyaguru, Michael Sangi, Ryan C. Schoenfeld, Leanna R. Staben,

Yunchou Tan, Joshua P. Taygerly, Armando G. Villasenor, and Paul E. Weller

̃

Pharma Research and Early Development, Hoﬀmann-La Roche Inc., 340 Kingsland Street, Nutley, New Jersey 07110, United States

S

* Supporting Information

ABSTRACT: In the past few years, there have been many

advances in the eﬀorts to cure patients with hepatitis C virus

(HCV). The ultimate goal of these eﬀorts is to develop a

combination therapy consisting of only direct-antiviral agents

(DAAs). In this paper, we discuss our eﬀorts that led to the

identiﬁcation of a bicyclic template with potent activity against

the NS5B polymerase, a critical enzyme on the life cycle of

HCV. In continuation of our exploration to improve the

stilbene series, the 3,5,6,8-tetrasubstituted quinoline core was identiﬁed as replacement of the stilbene moiety. 6-Methoxy-2(1H)-

pyridone was identiﬁed among several heterocyclic headgroups to have the best potency. Solubility of the template was improved

by replacing a planar aryl linker with a saturated pyrrolidine. Proﬁling of the most promising compounds led to the identiﬁcation

of quinoline 41 (RG7109), which was selected for advancement to clinical development.

convenient once daily dosing (q.d.) regimen than the ﬁrst two

compounds approved with three times daily (t.i.d.) and twice

daily (b.i.d.) dosing regimens, respectively.⁵The use of other

direct-acting antivirals with diﬀerent mechanisms of action

(polymerase nucleoside inhibitor (NI) and non-nucleoside

inhibitor (NNI) as well as nonstructural 5A (NS5A) inhibitors)

has shown SVR > 80% in combination with Peg-IFN/RBV in

phase II clinical studies.⁴

Interferon-associated side eﬀects limit treatment success in a

large number of patients.⁶Therefore, one of the primary future

goals is to develop direct-acting antiviral (DAA) therapy that

can achieve high SVR without the use of Peg-IFN. For this

objective, there are currently several clinical trials ongoing with

combinations of DAAs (protease inhibitor, polymerase NI,

polymerase NNI, and NS5A inhibitor) to determine which will

deliver the best results with respect to SVR and safety for

diﬀerent patient populations and against the most common

genotypes (GT-1, -2, -3, and -4).⁶The development of potent

and safe inhibitors of the diﬀerent viral proteins is crucial to be

able to achieve the goal of DAA therapy. The discovery of

INTRODUCTION

■

Hepatitis C virus (HCV), a positive-strand RNA virus member

of the Flaviviridae family, chronically infects 150 million people

worldwide with an estimated incidence of new cases of 3−4

million each year.¹HCV infection results in mild and acute liver

disease, but chronic infections are common and may eventually

develop into liver cirrhosis or hepatocellular carcinoma.²

Until 2011, the standard of care (SOC) for treating HCV had

been a combination therapy of pegylated interferon α (Peg-

IFN) plus ribavirin (RBV). Response rates for HCV patients

having genotype 2 or 3 on a 24−48 week treatment show a

sustained virological response (SVR, deﬁned as the absence of

detectable HCV RNA in blood serum for 24 weeks after

treatment withdrawal) approaching 80%. Patients infected with

HCV genotype 1 (GT-1) do not respond as well to this

combination therapy, demonstrating SVR rates of <50% even

after treatment therapies of 48 weeks in duration.³

With the recent approval of two HCV protease inhibitors

(telaprevir and boceprevir) by the FDA, the standard of care for

treatment of GT-1 infection is now Peg-IFN/RBV and a

protease inhibitor. This triple combination improves the SVR

rates up to 75% (telaprevir) and 68% (boceprevir) with

genotype-1 naive patients⁴and reduces treatment duration.

Other protease inhibitors in clinical development are showing

improved SVR rates of >90% in some patients and with a more

Special Issue: HCV Therapies

Received: August 28, 2013

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polymerase inhibitors and the development of a combination

therapy that would eventually replace interferon-based treat-

ment have been of interest to us for some time.^7,8

The HCV nonstructural 5B (NS5B) protein contains the

RNA-dependent RNA polymerase, the catalytic component of

the HCV RNA replication machinery. This virus-speciﬁc

enzyme plays an important role in the viral life cycle and

thus represents an attractive therapeutic target. Several classes

of inhibitors have been reported for the NS5B polymerase: NIs

that bind in the active site, and NNIs that bind in at least four

diﬀerent allosteric sites identiﬁed so far (palm I, palm II, thumb

I, and thumb II).⁹

Diﬀerent chemotypes have been reported to bind in the palm

I allosteric site. From those chemical classes, at least ﬁve

compounds have entered clinical trials and some of them have

shown eﬃcacy. Those are the N-acylpyrrolidine GSK625433

(1),¹⁰benzothiadiazine ANA598 (2),¹¹benzodiazaphosphinine

IDX375 (3),¹²and the uracil derivatives ABT-072 (4)¹³and

ABT-333 (5)¹⁴(Figure 1).

Figure 2. 3-Arylpyridones. Biological data were generated with the

HCV GT-1b strain.

(heterocyclic head and the sulfonamide group) in the right

position to interact with the critical amino acids in the binding

site. Among the diﬀerent possibilities to achieve this, the option

to have a bicyclic core that held both critical groups seemed to

ﬁt well based on modeling. Structure 8 was designed by

analyzing the cocrystal structure of an analogue of stilbene 7

with NS5B¹⁶where it was observed that the vinyl carbon linked

to the phenylsulfonamide could be bridged to C-6 of the central

core. Several advantages were foreseen by having a bicyclic

aromatic core, e.g., restricted rotation into bound conformation

for potency, opportunity to explore diﬀerent linker groups to

the sulfonamide, and use of heteroaromatic systems to

modulate its druglike properties. On the basis of this rationale,

it was decided to explore bicyclic aromatic cores.

In this article, we describe our eﬀorts on the exploration of

the bicyclic core that led to the discovery of RG7109 (41), a

potent inhibitor of the HCV NS5B polymerase that was

selected for clinical development.

CHEMISTRY

■

To determine if a bicyclic core would maintain either similar or

improved potency with respect to the stilbene series, four

diﬀerent ring systems were synthesized. Of the four bicyclic

cores reported in Table 1, the 3,5,6,8-tetrasubstituted quinoline

system was prepared using two similar routes as shown in

Schemes 1 and 2.

In Scheme 1, the use of 4-tert-butyl-3-methoxybenzoic acid

(9) as starting material allowed us to have two of the four

desired substitutions already in place. This starting material was

very useful, since we planned to maintain these two groups in

most of the initial set of bicyclic compounds. The ﬁrst step of

the synthesis was to convert the carboxylic acid 9 to the

primary aniline 11 by a Curtius rearrangement via the

corresponding carbamate 10. Formation of the desired

quinoline ring, using the amine group in 11 as a handle, had

the disadvantage of producing two possible regioisomers. To

avoid this, selective bromination of 11 para to the methoxy

group to aﬀord bromoaniline 12 was carried out prior to the

cyclization reaction. Treatment of compound 12 with 2,2,3-

tribromopropanal (prepared in situ from 2-bromoacrolein and

bromine) achieved the formation of the desired 3,8-

dibromoquinoline 13 ring system in moderate yield.¹⁷At this

point, compound 13 possessed the two handles needed to add

the required substitution on the template to complete the

Figure 1. Known HCV polymerase inhibitors that bind in the palm I

allosteric site.

We have been interested in the design of inhibitors that bind

to the palm I allosteric site. Recently, we reported the de novo

design of fragment 6¹⁵(Figure 2) with submicromolar potency

against the HCV NS5B polymerase and the development of

that fragment into stilbene 7, a potent inhibitor of NS5B with

single digit nanomolar activity in the HCV subgenomic replicon

assay.¹⁶From the start of our eﬀorts, the objective was to ﬁnd

highly potent inhibitors of the replication of HCV with similar

activity against both GT-1a and GT-1b. Even though 7 had an

excellent biological proﬁle, we continued exploring diﬀerent

scaﬀolds. It was important to keep the key functionalities

B

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substitution pattern is depicted in Scheme 2. For this alternative

route, attachment of the heterocyclic group was done before

the formation of the quinoline system. Scheme 1, being more

versatile by virtue of introducing one of the variable elements

(heterocyclic headgroup) after the modest-yielding quinoline-

forming cyclization step, was used for most of the examples.

The naphthalene core with the appropriate substitution

pattern was built starting from 7-bromotetralin-1-one (18),

which was transformed to the corresponding trimethylsilane

(TMS) enolate followed by alkylation to yield compound 19

(Scheme 3). Bromination at the α position of the ketone group

aﬀorded tetralin-1-one 20, which was subsequently aromatized,

and the resulting phenol was O-alkylated to obtain trisub-

stituted naphthalene 21. Introduction of the second bromine

para to the methoxy group was carried out as described above

to obtain compound 22. With the required substitution on the

naphthalene core in place, the 4-(methanesulfonamido)phenyl

and heterocycle groups were introduced to obtain the ﬁnal

product 24.

Table 1. Bicyclic Core Templates with Replicon (GT-1a and

GT-1b) Data

Synthesis of the 2,5,7,8-tetrasubstituted quinoline was

achieved by using a three-component imino Diels−Alder

reaction as reported by Kouznetsov.¹⁸Treatment of aniline

25 with 4-nitrobenzaldehyde followed by addition of the

dienophile and Lewis acid gave a mixture of tetrahydroquino-

lines and desired quinoline 26. The mixture of tetrahydroqui-

nolines was oxidized with ceric ammonium nitrate (CAN) to

aﬀord quinoline 26 in 22% overall yield. Compound 26 was

converted to the ﬁnal product by reduction of its nitro group

followed by methanesulfonylation of the resultant aniline and

addition of the heterocycle headgroup at the C-5 position via

cross-coupling (Scheme 4).

The last bicyclic template synthesized was the 2,4,7-

trisubstituted quinazoline as described in Scheme 5. 2-Amino-

4-bromobenzamide (29) was reacted with pivaloyl chloride

followed by thermal ring closure under alkaline conditions to

give corresponding quinazolin-4-one 30. Coupling of com-

pound 30 with 4-(methylsulfonamido)phenylboronic acid

followed by reaction with phosphorus oxychloride aﬀorded

the chloroquinazoline 31 in good yield. The desired ﬁnal

product 32 was obtained following standard cross-coupling

conditions for the addition of the heterocycle on the C-4

position.

RESULTS AND DISCUSSION

■

Compound 33, prepared according to Scheme 1, was made ﬁrst

to determine if the bicyclic core could replace the stilbene

moiety in our previous series and still maintain potency.

Compound 33 has the same 2(1H)-pyridone head as our lead

stilbene 7 to allow us to make a close comparison. For 5-

ﬂuoropyridone 33, the nitrogen of the quinoline system was

expected to face the vicinity of the backbone of Tyr448 and

Gly449, where a conserved molecule of water is normally

observed in the crystal structures. It was thought that a

hydrogen bond acceptor facing that area would be beneﬁcial.⁷

Compound 33 displayed single-digit nanomolar activity and

equipotency against both GT-1a and GT-1b strains in the

replicon assay (Table 1). An attractive advantage of 5-

ﬂuoropyridone 33 was that it had a slightly better activity

against GT-1a than stilbene 7. With this encouraging result, we

explored the possibility of removing the methoxy group from

the core to determine its eﬀect on potency. A 4- to 6-fold drop

in activity was observed with the desmethoxy analogue 34,

a

EC₅₀measured using stable HCV (GT-1a H77, GT-1b Con1)

subgenomic replicon assay (n ≥ 2).

synthesis. Coupling of compound 13 with the corresponding

boronic acid using Pd(PPh₃)₄occurred in a regioselective

manner at the least hindered bromine, aﬀording 3-substituted

quinoline 14. Under similar reaction conditions, the hetero-

cycles were attached onto C-8 of the quinoline system to give

the desired ﬁnal compounds. The second route utilized to

synthesize derivatives of the quinoline with this speciﬁc

C

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a

Scheme 1. Synthesis of 3,5,6,8-Tetrasubstituted Quinoline Core

a

Reagents and conditions: (a) Ph₂PON₃, Et₃N, EtOH, THF, reﬂux, 81%; (b) aq KOH, EtOH, reﬂux, 96%; (c) NBS, DMF, quantitative; (d) Br₂, 2-

bromoprop-2-enal, AcOH, 100 °C, 45%; (e) 4-(methanesulfonamido)phenylboronic acid, Pd(PPh₃)₄, Na₂CO₃, MeOH, toluene, 120 °C, 57%.

a

Scheme 2. Alternative Synthesis of 3,5,6,8-Tetrasubstituted Quinoline Core

a

Reagents and conditions: (a) (2-methoxy-3-pyridyl)boronic acid, Pd(PPh₃)₄, Na₂CO₃, MeOH, CH₂Cl₂, 115 °C, 54%; (b) Br₂, 2-bromoprop-2-enal,

AcOH, 100 °C, 27%.

a

Scheme 3. Synthesis of 2,5,7,8-Tetrasubstituted Naphthalene Core

a

Reagents and conditions: (a) TMSCl, NaI, Et₃N, CH₃CN, 69%; (b) 2-chloro-2-methylpropane, TiCl₄, CH₂Cl₂, −40 °C, 50%; (c) Br₂, AcOH, 50

°C, 98%; (d) LiBr, Li₂CO₃, DMF, 100 °C; (e) MeI, K₂CO₃, DMF, 96% two steps; (f) Br₂, AcOH, 98%; (g) 4-(methanesulfonamido)phenylboronic

acid, Pd(PPh₃)₄, Na₂CO₃, MeOH, toluene, 115 °C, 54%.

indicating that the bicyclic core required a methoxy group for

potency and overall proﬁle. For this exploration, 5-ﬂuoropyr-

idone head was replaced with the uracil heterocycle while

maintaining the methoxy, tert-butyl, and N-phenylmethanesul-

fonamide substituents. Uracil 35 displayed a decrease in activity

good potency.

After these initial results, we continued exploring diﬀerent

bicyclic cores to determine which would give us the best

D

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a

Scheme 4. Synthesis of 2,5,7,8-Tetrasubstituted Quinoline Core

a

Reagents and conditions: (a) 4-nitrobenzaldehyde, N-vinylpyrrolidin-2-one, BiCl₃, CH₃CN; (b) CAN, CH₃CN, 0 °C, 22% two steps; (c) Fe,

NH₄Cl, MeOH, water, reﬂux, 73%; (d) MsCl, pyridine, CH₂Cl₂, 0 °C, 86%.

With these initial results, we started our optimization eﬀorts

of the 3,5,6,8-tetrasubstituted quinoline core by exploring

diﬀerent heterocyclic head groups. Table 2 shows the

compounds made along with their activity in the replicon

assay. The ﬁrst three compounds after compound 35 have a

2(1H)-pyridone as a headgroup. Of the three, compound 41

stood out because of its excellent potency and equipotency

against both GT-1a and GT-1b. The dihydrouracil 42 displayed

good potency and selectivity comparable to those of uracil 35.

When the dihydrouracil head of 42 was replaced by a cyclic

urea moiety, the potency of the compound (43) decreased by

14-fold against GT-1a and 6-fold against GT-1b. A possible

explanation for the decrease in potency is the diﬀerence in pK_a

of the NH of the dihydrouracil with respect to that of the cyclic

urea (calculated¹⁹pK_afor dihydrouracil 42 is 8.7, and that of

cyclic urea 43 is 13.7). The increase in pK_acould decrease the

strength of the interaction of the NH with the backbone

carbonyl of Gln446, resulting in a decrease in potency. C-linked

uracil 44 displayed improved potency (∼2-fold) against both

genotypes with respect to N-linked uracil 35.

Scheme 5. Synthesis of 2,4,7-Trisubstituted Quinazoline

Core

a

The design of the bicyclic cores and head replacements was

based on our understanding of the binding mode of the stilbene

series in the palm I site.¹⁶The expected binding mode of the

bicyclic series was conﬁrmed from the crystal structure of

compound 41 bound to NS5B (Figure 3). A careful analysis of

the data shows that the 2(1H)-pyridone head is within

hydrogen bond distance to the backbone carbonyl of Gln446

and the NH of Tyr448 (2.9 Å in both cases). The lower ring of

the quinoline template makes edge-to-face interactions with the

side chain of Try448, and the tert-butyl group ﬁlls up the lower

lipophilic pocket. These interactions are the same as those

observed previously with the original fragment 6.¹⁵The

nitrogen of the quinoline core is within bonding distance to a

conserved molecule of water (not shown in Figure 3). The

main role for this ring, as replacement of the vinyl group, is to

hold the linker of the methanesulfonamide in the right position.

The phenyl ring that links the quinoline with the

methanesulfonamide makes an edge-to-face interaction with

Phe193, and the sulfonamide group is within hydrogen bond

distance from the residues Asp318, Asn 291, and Ser288 (2.7,

2.8, and 3.2 Å, respectively). The sulfonamide functional group

was essential for the high potency of the template, indicative of

the importance of these interactions. The oxygen of the

methoxy group on C-6 of the 2(1H)-pyridone is in proximity

(3.3 Å) to the carbon of the backbone carbonyl Gly410.

At this point in our eﬀorts to improve activity, the quinoline

series had shown excellent potency in the HCV replicon assay

against both GT-1a and GT-1b. Also, most of the

a

Reagents and conditions: (a) pivaloyl chloride, Et₃N, CH₂Cl₂; (b) aq

NaOH, EtOH, reﬂux, 52% two steps; (c) 4-(methanesulfonamido)-

phenylboronic acid, Pd(PPh₃)₄, Na₂CO₃, MeOH, toluene, 115 °C,

84%; (d) POCl₃, DIPEA, benzene, reﬂux, 87%.

against GT-1a but maintained the same potency against GT-1b

with respect to 5-ﬂuoropyridone 33 in the replicon assay. For

compounds 36−38, the uracil headgroup was held constant so

that the activity of diﬀerent bicyclic cores could be compared

with that of quinoline 35. Naphthalene 36 showed a 2- to 3-

fold increase in potency against both genotypes with respect to

quinoline 35. Moving the quinoline nitrogen of 35 to the

opposite position (37) caused the potency to decrease

signiﬁcantly. This drop in activity could be partially explained

by the expected almost planar orientation of the N-

phenylmethanesulfonamide group with respect to the quinoline

ring due to the introduction of the quinoline nitrogen adjacent

to the phenyl ring. Finally, quinazoline 38 was screened only

against GT-1a and also displayed lower activity than quinoline

35.

This initial exploration showed that the naphthalene core

(36) had the best potency proﬁle among the four bicyclic cores.

Even though the naphthalene core presented the best activity,

the 3,5,6,8-tetrasubstituted quinoline core was selected to

continue exploring modiﬁcations on other parts of the template

because of its ease of synthesis, ﬂexibility to make

modiﬁcations, and excellent potency in the replicon assay.

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Table 2. Diﬀerent Headgroups on the 3,5,6,8-

Tetrasubstituted Quinoline Core with Replicon (GT-1a and

GT-1b) Data

Figure 3. Cocrystal structure of compound 41 with NS5B polymerase

(GT-1b BK) in palm I allosteric site. Crystal structure ﬁgures were

drawn with PyMOL.²⁰

Table 3. Replacements of the Phenyl Linker with NS5B (GT-

1b) and Replicon (GT-1a) Data

a

EC₅₀measured using stable HCV (GT-1a H77, GT-1b Con1)

subgenomic replicon assay (n ≥ 2).

physicochemical and ADME in vitro properties were within the

desirable range (e.g., microsomal stability, CYP inhibition,

permeability) except for solubility. Some of the compounds

with the best biological activity exhibited low aqueous solubility

(<5 μg/mL). One approach to improve this property was by

reducing the planarity of the compounds. With this in mind, we

explored diﬀerent replacements of the phenyl linker between

the quinoline and the methanesulfonamide (Table 3). The

four-carbon alkyl or alkyne linkers (45 and 46, respectively)

exhibited activity in the enzyme assay, but it was not transferred

to the replicon assay. The weak activity observed for these two

compounds could be attributed to the diﬀerent possible

conformations of the linker which would prevent the

methanesulfonamide group from making favorable interactions

a

IC₅₀values against NS5B enzyme were measured using GT-1b Con1

b

strain and reported in nM. EC₅₀measured using stable HCV GT-1a

H77 subgenomic replicon assay (n ≥ 2). Aqueous solubility was

measured using a thermodynamic solubility assay.

c

with Asp318, Asn 291, and Ser288. In order to minimize the

number of possible conformations, we explored saturated ring

systems as linkers. Azetidine (47) and piperidine (49) linkers

gave similar results as the alkyl chains. To the contrary, the

pyrrolidine ring linker (48) displayed the same potency as the

corresponding phenyl linker (39). These results were expected,

since the conformationally restricted ring systems of 47−49

would place the methanesulfonamide group in slightly diﬀerent

F

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positions, resulting in diﬀerent activities. Compound 48 was

cocrystallized with the NS5B polymerase, and it demonstrates

in the palm I site the same interactions as described for

quinoline 41. The pyrrolidine ring is within van der Waals

radius contact distance of Phe193 (3.9−4.2 Å) and places the

methanesulfonamide in the right position to make the same

network interactions in the catalytic site as quinoline 41 (Figure

4).

dosing regimen in the clinic. Even though the majority of the

compounds screened in our in vitro hepatocyte assay had

medium to high stability (data not shown), a focused eﬀort was

carried out to determine if this parameter could be improved.

As we showed in the stilbene template,¹⁶oxidation of the tert-

butyl group in the bicyclic template was one of the major routes

of metabolism (data not shown). On the basis of this

observation, replacements of the tert-butyl were made on the

uracil 35 template to determine if it was possible to improve the

stability. Replacement of the methyl groups on the tert-butyl by

ﬂuorines (triﬂuoromethyl 51) led to improved stability in

human and rat hepatocytes, but a large drop in activity was

observed against both GT-1a and GT-1b (Table 4). These

Table 4. Replacements of tert-Butyl Group with Replicon

(GT-1a and GT-1b) and in Vitro Hepatocytes Data

Figure 4. Cocrystal structure of compound 48 with NS5B (GT-1b

BK) polymerase in palm I allosteric site.

As anticipated, the replacement of the phenyl ring with a

saturated linker (in this case the pyrrolidine ring) led to an

increase in aqueous solubility (Table 3). The combination of

good potency and improved solubility made the pyrrolidine

linker an attractive option for further exploration. The phenyl

linker of the best compounds described in Tables 1 and 2 was

replaced with the pyrrolidine linker. The replacements also led

to an increase in aqueous solubility, but potency was not always

maintained in comparison to the corresponding phenyl linker

compounds (data not shown). From this set of compounds,

pyridone 50, the pyrrolidine analogue of compound 41, had the

best combination of overall properties (Figure 5). Although the

potency of pyrrolidine 50 in the replicon assay was weaker than

compound 41, it still maintained almost equal potency against

both GT-1a and GT-1b and good aqueous solubility.

a

EC₅₀measured using stable HCV (GT-1a H77 and GT-1b Con1)

b

subgenomic replicon assay (n ≥ 2). Human hepatocyte (HH) and rat

hepatocyte (RH) intrinsic clearance is reported in (μL/min)/10⁶cells.

Human hepatocyte category ranking: high stability, <2.8; low stability,

>15. Rat hepatocyte category ranking: high stability, <5.4; low stability,

>29.

It was desired to select a compound with a long half-life in

vivo to be able to dose the compound with a q.d. or b.i.d.

results supported our assumption that the metabolism of tert-

butyl group was responsible for most of the clearance observed.

Two additional modiﬁcations were made to determine if

cellular activity could be maintained while improving metabolic

stability. Extension of the triﬂuoromethyl group by one carbon

(1,1,1-trifuoroethyl 52) had a similar outcome as with

triﬂuoromethyl 51. The loss of potency seen in compounds

51 and 52 could be due to the less than optimal ﬁlling of the

lower lipophilic pocket in the palm I site. To address this

observation, compound 53 was prepared that contains a

cyclopropyl group instead of two of the methyl groups of the

tert-butyl and a diﬂuoromethyl as replacement for the third

methyl. These two changes maintain similar ﬁlling of the pocket

as the tert-butyl but with metabolically stable substitutions.

Cyclopropyl 53 displayed the expected high metabolic stability

with an improvement in potency of 3- to 9-fold with respect to

compounds 51 and 52 but still weaker than uracil 35. Similar

Figure 5. Best pyrrolidine derivative from the bicyclic series.

G

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Table 5. Activity in the Replicon Assay for a Selected Set of Compounds in the Bicyclic Series

a

EC₅₀(nM)

EC₉₀(nM)

b

compd

GT-1a

GT-1b

ratio 1a/1b

GT-1a, 40% HuS

GT-1b, 40% HuS

plasma shift fold

GT-1a, 40% HuS

GT-1b, 40% HuS

7

8.0

7.2

5.2

1.1

4.6

17

3.0

3.2

3.1

1.0

3.2

12

2.7

2.2

1.7

1.1

1.4

66

16

4.0

10

22

7.3−8.2

1.7−2.2

0.8−3.5

3.8−9.4

1.7−2.1

0.6

531

123

25

128

29

47

19

20

44

35

39

41

42

50

5.6

11

3.8

3.5

7.5

63

48

61

a

b

EC₅₀and EC₉₀measured using stable HCV (GT-1a H77, GT-1b Con1) subgenomic replicon assay (n ≥ 2). Human serum (HuS).

tert-butyl replacements have been recently reported with similar

improvements in in vitro stability.²¹Unfortunately, the overall

proﬁle of these tert-butyl replacements did not meet our

objective, and they were not pursued further.

Table 6. Activity of 41 and 50 against GT-1a and GT-1b

Clinical Isolates

a

EC₅₀SEM (nM)

GT

1a

clinical isolate

41

50

After exploration of the four diﬀerent substitutions of the

bicyclic template and an understanding of their role, ﬁve

compounds were selected for further proﬁling in the replicon

assay (Table 5). All the compounds selected showed potent

inhibition against both GT-1a and GT-1b with almost

equipotency against both genotypes (ratio 1a/1b = 1.1−2.2).

The compounds were screened in the replicon assay in the

presence of 40% human serum (HuS), a more relevant

measurement of activity in terms of prediction of clinical

eﬃcacy. All the compounds maintained potency at or below 10

nM under the conditions of this assay. The plasma shift

observed was relatively small (0.6- to 3.5-fold) for all the

compounds except for pyridone 41 (3.8- to 9.4-fold). The EC₉₀

in the presence of 40% HuS was used to determine the plasma

trough concentration (C_min) needed to obtain eﬃcacy in the

clinic. We targeted potency at 5-fold coverage above the

measured EC₉₀. Since this series generally had a weaker potency

against GT-1a in the presence of 40% HuS, we decided to use

this value to predict eﬃcacy in human.

From this set of compounds, 41 and 50 were selected for

further biological proﬁling against a panel of GT-1a and GT-1b

NS5B clinical isolates from untreated patients in a transient

replicon assay.²²Both compounds showed similar or improved

potency against the clinical isolates (transient replicon, Table 6)

compared to the potencies observed using the stable HCV

replicons (Table 5). Diﬀerences between the two HCV

replicon systems have been previously reported²³and could

be due to a better inhibition of the HCV replicase complex in

the transient replicon by the non-nucleoside inhibitors than in

the stable replicon system. The major loss in potency in

comparison with the rest of the GT-1a panel was seen with

clinical isolate RO-38. The sequence of this NS5B isolate

contains mutation S556G which is located close to the catalytic

site where both of our compounds bind. The mean EC₅₀for

compounds 41 and 50 in this set of clinical isolates was shown

to be very similar. Against the panel of GT-1b clinical isolates,

compound 41 displayed consistent values against the isolates

and a mean EC₅₀similar to the one observed with the stable

line. Compound 50 displayed a slight range of activities across

the panel but still with a low single-digit nanomolar mean EC₅₀

value.

H77

0.3 0.03

0.4 0.05

1.1 0.23

0.9 0.05

0.6 0.09

0.5 0.02

0.3 0.09

4.3 0.07

0.6 0.17

0.72

0.65 0.12

0.5 0.26

2.5 0.41

0.2 0.08

0.7 0.03

0.4 0.08

0.5 0.08

9.5 0.41

0.27 0.07

0.8

RO-18

RO-24

RO-28

RO-33

RO-34

RO-35

RO-38

RO-41

mean

1b

Con1

RO-1

RO-2

RO-3

RO-4

RO-7

RO-8

RO-9

RO-10

mean

0.32 0.08

0.11 0.01

0.19 0.003

0.43 0.02

0.11 0.003

0.22 0.02

0.33 0.10

0.34 0.04

0.34 0.03

0.26

1.20 0.27

0.30 0.06

4.00 1.63

6.1 0.12

0.80 0.16

3.05 0.37

0.33 0.10

3.50 0.41

1.67 0.33

1.6

a

EC₅₀measured using transient HCV subgenomic replicon assay (n ≥

2), listed with SEM (standard error mean). Geometric mean was

calculated.

our criteria to enable b.i.d. dosing in humans (Table 7).

Bioavailability was moderate in both species, achieving good

levels at C_max. In addition, concentrations of 0.62 μg/mL (1.22

μM) and 0.99 μg/mL (1.95 μM) in rat and dog, respectively,

were observed at 8 h after oral dosing. These concentrations

cover over 19-fold the EC₉₀observed for 41 in the GT-1a

replicon assay in the presence of 40% human serum. These

pharmacokinetic data together with the biological proﬁle

described above lent additional conﬁdence that suppression

of the virus in humans could be achieved with compound 41

based on our prediction model.²⁶It was estimated that 41 will

show a viral load reduction of >1.5 in a 3-day monotherapy

with a dose of 49−120 mg b.i.d.

Compound 50 displayed a good proﬁle in both species

(Table 8). When dosed to rats, it showed low clearance and a

slightly better t_1/2than 41 with an excellent bioavailability.

These characteristics allowed compound 50 to reach a

concentration of 1.79 μg/mL (3.5 μM) at 8 h after oral

dosing. This concentration covers over 50-fold the EC₉₀

observed for 50 in the GT-1a replicon assay in the presence

of 40% human serum. The pharmacokinetic parameters in dog

for 50 were similar to those observed in rat.

The pharmacokinetic properties of compounds 41 and 50

were determined in rat and dog. Because of the low solubility of

41 (aqueous solubility of <1 μg/mL), this compound was

dosed as a microprecipitated bulk powder (MBP) for oral

dosing as described by Shah et al.^24,25Compound 41 showed

low clearance in both rat and dog with a t_1/2of 4.3 h, which met

H

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Table 7. Pharmacokinetic Parameters for 41 in Rat and Dog

iv

po

a

b

species

dose

Cl (mL min⁻¹kg⁻¹

)

V_dss(L/kg)

t_1/2(h)

dose

C_max(μg/mL)

C at 8 h (μg/mL)

AUC(inf) (μg·h/mL)

F (%)

rat

2.6

11.4

7.5

2.2

2.8

4.3

30

2.1

1.5

0.62

0.99

13.8

23.7

26

31

dog

0.86

a

b

Dose in mg/kg, 20% PEG400, n = 3. Dose in mg/kg, amorphous MBP, n = 3.

Table 8. Pharmacokinetic Parameters for 50 in Rat and Dog

iv

po

a

b

species

dose

Cl (mL min⁻¹kg⁻¹

)

V_dss(L/kg)

t_1/2(h)

dose

C_max(μg/mL)

C at 8 h (μg/mL)

AUC(inf) (μg·h/mL)

F (%)

rat

3.0

1.0

13.8

4.4

2.3

1.5

5.6

5.1

30

3

3.8

1.79

0.21

37.0

4.2

100

36

dog

0.36

a

b

Dose in mg/kg, 20% PEG400, n = 3. Dose in mg/kg, aqueous suspension, n = 3.

Sigma-Aldrich in a Sure/Seal system. Puriﬁcation of crude material

was performed using AnaLogix IntelliFlash 280 or Teledyne Isco

CombiFlash Companion system using silica gel columns RediSep Rf

(particle size 40−63 μm powder). Deuterated solvents were purchased

from Sigma-Aldrich. Proton nuclear magnetic resonance (¹H NMR)

spectra were recorded on a Bruker Avance 300, 400, or 500 MHz

spectrometer at 25 °C. Chemical shifts for protons are reported in

parts per million downﬁeld from tetramethylsilane and are referenced

to residual protium in the NMR solvent (CHCl₃δ 7.27, DMSO δ

2.50). Data are represented as follows: chemical shift, multiplicity (br

= broad, s = singlet, d = doublet, t = triplet, q = quartet, m =

multiplet), coupling constants in hertz (Hz), and integration. High

resolution mass spectrometry (HRMS) data were obtained on a

Thermo Finnigan LTQ Orbitrap XL spectrometer operated in ESI

mode at a resolution of 30 000. Liquid chromatography−mass

spectrometry (LC/MS) analyses were carried out using an Agilent

6140 single quadrupole in ES/APCI dual mode and Agilent LC with a

Zorbax SB-C18 2.1 mm × 30 mm, 3.5 μm column using a 0.02%

TFA/water−acetonitrile solvent system. Microwave reactions were

carried out in a Biotage initiator reactor. Purity of tested compounds

was ≥95% as conﬁrmed by LC/MS.

Compounds 41 and 50 both possess overall characteristics

desirable for a clinical candidate. Compound 41 (RO5471354)

was ultimately selected for advancement to clinical develop-

ment.

CONCLUSION

■

In summary, exploration of a diverse set of bicyclic cores to

replace our previous stilbene core led us to the identiﬁcation of

the 3,5,6,8-tetrasubstituted quinolines. This series displayed

excellent potency in the HCV replicon assay and a minimal

separation in activity between GT-1a and GT-1b. Optimization

of the headgroup steered us to the identiﬁcation of the 6-

methoxypyridone as one of the most promising heads due to its

potency and equal activity against both GT-1a and GT-1b.

Replacement of the phenyl linker for saturated alkyl and

cycloalkyl groups identiﬁed 3-substituted pyrrolidine as a

promising linker. This linker maintained the potency and

improved the solubility of the template. Eﬀorts to replace the

tert-butyl for a more metabolically stable group were successful

in improving the stability of the template, but the replacements

did not maintain the desired potency in the replicon assay.

Overall, the optimization eﬀorts led us to select ﬁve compounds

for further biological proﬁling. Compounds 41 and 50 were

then selected for pharmacokinetic studies in rat and dog to

determine their potential as clinical candidates. Both com-

pounds met or exceeded our criteria for clinical advancement

based on the results described above. Ultimately, compound 41

was chosen for clinical development.

Ethyl N-(4-tert-Butyl-3-methoxyphenyl)carbamate (10). To a

solution of 4-tert-butyl-3-methoxybenzoic acid (9) (10.4 g, 50.0 mmol)

in THF (200 mL) was added diphenylphosphorylazide (11.3 mL, 52.4

mmol), Et₃N (8.4 mL, 60.3 mmol), and EtOH (29 mL) at room

temperature. The mixture was heated at reﬂux for 23 h, cooled to

room temperature, and concentrated. The residue was dissolved in

EtOAc (300 mL), washed with saturated aqueous NaHCO₃, dried

over MgSO₄, and evaporated. The crude material was puriﬁed over

SiO₂(gradient, 0−25% EtOAc in hexane) to give ethyl carbamate 10

1

(10.12 g, 81%) as an oﬀ-white solid. H NMR (400 MHz, CDCl₃) δ

7.16 (d, J = 8.6 Hz, 1 H), 6.68 (dd, J = 8.2, 2.9 Hz, 1 H), 6.52 (br s, 1

H), 4.22 (q, J = 7.0 Hz, 2 H), 3.84 (s, 3H), 1.35 (s, 9 H), 1.31 (t, J =

7.3 Hz, 3 H). LC/MS (ES/APCI): 252.1 (M + H)⁺.

Our eﬀorts to explore diﬀerent scaﬀolds to improve the

proﬁle of our lead compound 7 yielded a compound with better

biological activity. Compound 41 is equipotent against the two

GTs and has a 6-fold improvement in the 40% HuS replicon

assay (Table 5).

The development of fragment 6 into a compound that was

selected for clinical development is another successful case

where the use of fragments¹⁵together with structure-based

design¹⁶led to the selection of a clinical candidate.

4-tert-Butyl-3-methoxyaniline (11). To a solution of ethyl

carbamate 10 (11.88 g, 47.3 mmol) in EtOH (86 mL) was added a

solution of KOH (29.17 g, 520 mmol) in EtOH (172 mL) at room

temperature. The reaction mixture was heated at reﬂux for 15 h and

then cooled to room temperature and concentrated. The residue was

partitioned with EtOAc (400 mL) and brine (400 mL). The organic

layer was separated, dried over MgSO₄, and concentrated to dryness.

The residue was puriﬁed over SiO₂(gradient, 0−35% EtOAc in

hexane) to give aniline 11 (8.16 g, 96%) as a yellow oil. ¹H NMR (400

MHz, CDCl₃) δ 7.10 (dd, J = 7.2, 2.2 Hz, 1 H), 6.42−6.33 (m, 2 H),

3.82 (s, 3H), 1.35 (s, 9 H). LC/MS (ES/APCI): 180.17 (M + H)⁺.

2-Bromo-4-tert-butyl-5-methoxyphenylamine (12). NBS (8.1

g, 45.5 mmol) was added to a solution of aniline 11 (8.16 g, 45.5

mmol) in DMF (150 mL) at room temperature. The reaction mixture

was stirred for 5 h at room temperature, diluted with EtOAc/hexane

(1:1, 300 mL), washed with 1 N NaOH (100 mL), brine (300 mL),

dried over MgSO₄, ﬁltered, and concentrated to aﬀord crude

bromoaniline 12 (11.75 g, quantitative) as brownish oil after drying

EXPERIMENTAL SECTION

■

General Information. All reactions were performed in round-

bottom ﬂasks. The ﬂasks were ﬁtted with rubber septa, and reactions

were conducted under inert atmosphere. Plastic syringes and stainless

steel needles were used to transfer air- and moisture-sensitive liquids.

Most commercial reagents were purchased from Sigma Aldrich, Alfa

Aesar, Strem, Lancaster, AstaTech, or TCI and used as received.

Solvents were purchased from Mallinckrodt and anhydrous

dimethylformamide (DMF) and tetrahydrofuran (THF) from

I

dx.doi.org/10.1021/jm401329s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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under high vacuum. ¹H NMR (400 MHz, CDCl₃) δ 7.24 (s, 1 H), 6.40

(br s, 1 H), 3.78 (s, 3H), 1.31 (s, 9 H). LC/MS (ES): 258.04 (M +

H)⁺.

diﬀerent layers were separated and the top layer (pentane) was dried

over MgSO₄, ﬁltered, and concentrated to give a yellow oil. The

residue was puriﬁed over SiO₂(hexane) to give pure (7-bromo-3,4-

dihydronaphthalen-1-yl)oxytrimethylsilane (9.1 g, 69%). ¹H NMR

(300 MHz, CDCl₃) δ 7.51 (d, J = 1.9 Hz, 1 H), 7.26 (dd, J = 7.9, 2.3

Hz, 1 H), 6.97 (d, J = 7.9 Hz, 1 H), 5.21 (t, J = 4.9 Hz, 1 H), 2.70 (t, J

= 7.9 Hz, 2 H), 2.31 (ddd, J = 12.8, 8.7, 4.9 Hz, 2 H), 0.26 (s, 9H).

3,8-Dibromo-6-tert-butyl-5-methoxyquinoline (13). A solu-

tion of Br₂(168 mg, 1.05 mmol) in AcOH (1.3 mL) was added to 2-

bromoprop-2-enal (148 mg, 1.1 mmol) in AcOH (1.5 mL) at room

temperature. After the mixture was stirred for 15 min, a solution of

bromoaniline 12 (260 mg, 1.01 mmol) in AcOH (5 mL) was added to

the reaction. The reaction mixture was heated at 100 °C for 4 h. The

reaction mixture was carefully poured into a cold saturated aqueous

NaHCO₃and then extracted with EtOAc. The organic layer was

washed with brine, dried over MgSO₄, ﬁltered, and concentrated under

vacuum. The crude residue was puriﬁed over SiO₂(gradient, 0−10%

EtOAc in hexane) to aﬀord dibromide 13 (170 mg, 45%) as a

brownish solid. ¹H NMR (400 MHz, CDCl₃) δ 8.94 (d, J = 2.6 Hz, 1

H), 8.53 (d, J = 1.9 Hz, 1 H), 8.08 (s, 1 H), 3.95 (s, 3 H), 1.49 (s, 9

H). LC/MS (ES/APCI): 374.0 (M + H)⁺.

A

solution of (7-bromo-3,4-dihydronaphthalen-1-yl)-

oxytrimethylsilane (6.85 g, 23.0 mmol) in CH₂Cl₂(23 mL) was

cooled to −40 °C, and 2-chloro-2-methylpropane (1.32 mL, 12.0

mmol) was added under N₂. A solution of TiCl₄(1.27 mL, 11.6

mmol) in CH₂Cl₂(6 mL) at −40 °C was added to the above solution

dropwise, while the reaction mixture was stirred vigorously to a dark

brown/purple suspension. The mixture was allowed to reach room

temperature and stirred over 3 days. The reaction mixture was poured

over ice, diluted with EtOAc and water, neutralized with saturated

aqueous NaHCO₃, and ﬁltered over a plug of Celite to remove chunky

white precipitate. The organic layer was separated, washed with brine,

dried over MgSO₄, ﬁltered, and concentrated. The residue was puriﬁed

over SiO₂(5/95, EtOAc/hexane) to yield tert-butyltetralin-1-one 19

N-[4-(8-Bromo-6-tert-butyl-5-methoxy-3-quinolyl)phenyl]-

methanesulfonamide (14). A sealed vial containing dibromide 13

(850 mg, 2.27 mmol), 4-(methanesulfonamido)phenylboronic acid

(539 mg, 2.5 mmol), Pd(PPh₃)₄(263 mg, 0.227 mmol), and Na₂CO₃

(725 mg, 6.83 mmol) in a mixture of MeOH (8 mL) and toluene (5

mL) was irradiated in a microwave reactor at 120 °C for 1 h. The

reaction mixture was cooled to room temperature and diluted with

EtOAc. The organic layer was washed with water, dried over MgSO₄,

ﬁltered, and concentrated under vacuum. The crude residue was

puriﬁed over SiO₂(gradient, 0−70% EtOAc in hexane) to aﬀord 8-

1

(3.26 g, 50%) as a yellow solid. H NMR (300 MHz, CDCl₃) δ 8.06

(d, J = 2.1 Hz, 1 H), 7.51 (dd, J = 8.2, 2.1 Hz, 1 H), 7.09 (d, J = 8.6

Hz, 1 H), 3.03−2.81 (m, 2 H), 2.34−2.23 (m, 2 H), 2.0−1.83 (m, 1

H), 1.09 (s, 9 H). LC/MS (ES/APCI): 281.1 (M + H)⁺.

2,7-Dibromo-2-tert-butyltetralin-1-one (20). To a solution of

tert-butyltetralin-1-one 19 (3.0 g, 10.7 mmol) in AcOH (40 mL) was

added a solution of Br₂(0.6 mL, 11.6 mmol) in AcOH (20 mL)

dropwise over 20 min via cannula to give a dark orange suspension.

The mixture was stirred at room temperature for 1 h and then warmed

to 50 °C and stirred for 1 h. An extra quantity of neat Br₂(0.1 mL, 1.9

mmol) was added and continued heating at 50 °C for another 1.5 h.

The mixture was poured over ice, diluted with EtOAc and water, and

neutralized with saturated aqueous NaHCO₃. The organic layer was

separated, washed with brine, dried over MgSO₄, ﬁltered, and

concentrated. The crude product was puriﬁed over SiO₂(hexane) to

1

bromoquinoline 14 (600 mg, 57%) as an oﬀ-white solid. H NMR

(400 MHz, CDCl₃) δ 9.20 (d, J = 2.0 Hz, 1 H), 8.50 (d, J = 2.0 Hz, 1

H), 8.08 (s, 1 H), 7.73 (d, J = 8.1 Hz, 2 H), 7.40 (d, J = 7.7 Hz, 2 H),

6.50 (br s, 1 H), 4.00 (s, 3 H), 3.11 (s, 3H), 1.52 (s, 9 H). LC/MS

(ES): 462.99 (M + H)⁺.

4-tert-Butyl-5-methoxy-2-(2-methoxy-3-pyridyl)aniline (16).

A sealed vial containing bromoaniline 12 (700 mg, 2.71 mmol), (2-

methoxy-3-pyridyl)boronic acid (612 mg, 4.4 mmol), Pd(PPh₃)₄(231

mg, 0.2 mmol), and Na₂CO₃(636 mg, 6 mmol) in a mixture of

MeOH (1 mL) and CH₂Cl₂(9 mL) was irradiated in a microwave

reactor at 115 °C for 1 h. The reaction mixture was cooled to room

temperature and diluted with EtOAc. The organic layer was washed

with water, dried over MgSO₄, ﬁltered, and concentrated. The crude

residue was puriﬁed over SiO₂(gradient, 0−50% EtOAc in hexane) to

1

obtain compound 20 (3.8 g, 98%). H NMR (300 MHz, CDCl₃) δ

8.18 (d, J = 2.1 Hz, 1 H), 7.58 (dd, J = 8.5, 2.1 Hz, 1 H), 7.13 (d, J =

8.1 Hz, 1 H), 3.24 (ddd, J = 17.8, 12.7, 5.1 Hz, 1 H), 2.85 (ddd, J =

17.4, 5.1, 2.5 Hz, 1 H), 2.55 (ddd, J = 14.9, 4.7, 3.0 Hz, 1 H), 2.29

(ddd, J = 17.0, 11.5, 4.7 Hz, 1 H), 1.31 (s, 9 H).

7-Bromo-2-tert-butyl-1-methoxynaphthalene (21). A white

suspension of compound 20 (3.8 g, 10.6 mmol), LiBr (275 mg, 3.17

mmol), and Li₂CO₃(780 mg, 10.6 mmol) in DMF (44.0 mL) was

bubbled with argon for 10 min. The mixture was heated at 100 °C

under N₂for 1 h and then cooled to room temperature. The mixture

was diluted with EtOAc, washed with water, brine, dried over MgSO₄,

ﬁltered, and concentrated to obtain 7-bromo-2-tert-butylnaphthalen-1-

ol (3.0 g) as a light brown viscous oil in pure form and used in the next

reaction without puriﬁcation. ¹H NMR (300 MHz, CDCl₃) δ 8.27 (br

s, 1 H), 7.64 (d, J = 9.4 Hz, 1 H), 7.49 (dd, J = 8.6, 2.4 Hz, 1 H), 7.23

(d, J = 8.2 Hz, 1 H), 7.37 (d, J = 9.0 Hz, 1 H), 1.52 (s, 9H).

To a suspension of 7-bromo-2-tert-butylnaphthalen-1-ol (2.9 g, 10.4

mmol) and K₂CO₃(3.59 g, 26.0 mmol) in DMF (29.7 mL) was added

MeI (0.78 mL, 12.5 mmol) and the mixture stirred at 25 °C for 18 h.

The mixture was diluted with EtOAc and water and neutralized with 1

N HCl. The organic layer was separated, washed with brine, dried over

MgSO₄, ﬁltered, and concentrated to obtain methoxynaphthalene 21

(3.0 g, 96% two steps) as a solid. ¹H NMR (300 MHz, CDCl₃) δ 8.22

(d, J = 1.7 Hz, 1 H), 7.67 (d, J = 8.6 Hz, 1 H), 7.53−7.45 (m, 3 H),

3.95 (s, 3 H), 1.49 (s, 9 H).

4,7-Dibromo-2-tert-butyl-1-methoxynaphthalene (22). To a

solution of methoxynaphthalene 21 (1.6 g, 5.46 mmol) in AcOH (30

mL) was added a solution of Br₂(0.28 mL, 5.46 mmol) in AcOH (20

mL) dropwise via addition funnel under N₂. The reaction mixture was

stirred at room temperature for 18 h and then diluted with EtOAc and

water and neutralized with saturated aqueous NaHCO₃. The organic

layer was separated, washed with brine, dried over MgSO₄, ﬁltered, and

concentrated to obtain compound 22 (2.0 g, 98%) in pure form as an

oil. ¹H NMR (300 MHz, CDCl₃) δ 8.22 (d, J = 1.8 Hz, 1 H), 8.02 (d, J

1

aﬀord aniline 16 (420 mg, 54%) as a brownish oil. H NMR (400

MHz, CDCl₃) δ 8.17 (dd, J = 4.7, 1.4 Hz, 1 H), 7.57 (dd, J = 6.8, 1.6

Hz, 1 H), 6.98 (dd, J = 7.1, 4.6 Hz, 1 H), 6.96 (s, 1 H), 6.32 (s, 1 H),

3.99 (s, 3 H), 3.83 (s, 3 H), 3.72−3.64 (br s, 2 H), 1.34 (s, 9 H). LC/

MS (ES): 287.13 (M + H)⁺.

3-(3-Bromo-6-tert-butyl-5-methoxy-8-quinolyl)-1H-pyridin-

2-one (17). A solution of Br₂(191 mg, 1.2 mmol) in AcOH (5 mL)

was added to 2-bromoprop-2-enal (230 mg, 1.7 mmol) in AcOH (5

mL) at room temperature. The solution was titrated to the appearance

of a faint reddish color. After the mixture was stirred at room

temperature for 15 min, a solution of aniline 16 (420 mg, 1.47 mmol)

in AcOH (5 mL) was added. The reaction mixture was heated at 100

°C for 2 h. The reaction mixture was carefully poured into a cold

saturated aqueous NaHCO₃and then extracted with EtOAc. The

organic layer was washed with brine, dried over MgSO₄, ﬁltered, and

concentrated. The crude residue was puriﬁed over SiO₂(1:1, EtOAc/

hexane) to aﬀord the corresponding 3-bromoquinoline 17 (155 mg,

27%) as a brownish oil. ¹H NMR (400 MHz, CDCl₃) δ 8.82 (d, J = 2.3

Hz, 1 H), 8.55 (d, J = 1.7 Hz, 1 H), 7.88 (s, 1 H), 7.62 (dd, J = 6.6, 1.5

Hz, 1 H), 7.35 (dd, J = 6.4, 1.8 Hz, 1 H), 6.38 (t, J = 6.9 Hz, 1 H), 3.99

(s, 3 H), 1.52 (s, 9 H). LC/MS (ES): 388.94 (M + H)⁺.

7-Bromo-2-tert-butyltetralin-1-one (19). To a suspension of 7-

bromotetralin-1-one (18) (10.0 g, 44.4 mmol) in CH₃CN (10 mL)

was added Et₃N (7.43 mL, 53.3 mmol) followed by chloro(trimethyl)-

silane (6.81 mL, 53.6 mmol) at room temperature under argon. A

solution of NaI (7.99 g, 53.3 mmol) in CH₃CN (72 mL) was added

dropwise to the white suspension via cannula and stirred at room

temperature for 30 min. The reaction mixture was diluted with ice-cold

pentane (200 mL) and then poured over ice−water. The three

J

dx.doi.org/10.1021/jm401329s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

= 9.1 Hz, 1 H), 7.79 (s, 1 H), 7.59 (dd, J = 8.7, 1.8 Hz, 1 H), 3.94 (s, 3

H), 1.47 (s, 9 H).

and the solution was stirred for 2 h at the same temperature and then

allowed to reach room temperature and stirred for additional 2 h. The

mixture was concentrated under vacuum, and the crude material was

used in the next reaction without puriﬁcation. To a suspension of the

crude material in EtOH (28 mL) was added a solution of 10 M NaOH

(2.8 mL, 28 mmol) and the resulting mixture reﬂuxed under N₂for 1.5

h. The mixture was poured onto ice, neutralized with 1 N HCl, and

extracted with EtOAc. The organic layer was washed with brine, dried

over MgSO₄, ﬁltered, and concentrated to quinazolinone 30 (2.07 g,

53% two steps) as a light yellow solid. ¹H NMR (300 MHz, CDCl₃) δ

10.21 (br s, 1 H), 8.10 (d, J = 8.4 Hz, 1 H), 7.92 (d, J = 1.7 Hz, 1 H),

7.56 (dd, J = 8.4, 1.5 Hz, 1 H), 1.45 (s, 9 H). LC/MS (ES/APCI):

281.0 [M + H]⁺.

N-[4-(5-Bromo-7-tert-butyl-8-methoxy-2-naphthyl)phenyl]-

methanesulfonamide (23). A sealed vial containing compound 22

(1.0 g, 2.69 mmol), 4-(methanesulfonamido)phenylboronic acid (578

mg, 2.69 mmol), Pd(PPh₃)₄(155 mg, 0.134 mmol), and Na₂CO₃(855

mg, 8.06 mmol) in MeOH (7.14 mL), toluene (3.57 mL), and water

(1.79 mL) was irradiated in a microwave reactor at 115 °C for 1.5 h.

The reaction mixture was diluted with EtOAc and water and was

neutralized with 1 N HCl. The organic layer was separated, washed

with brine, dried over MgSO₄, ﬁltered, and concentrated. The crude

product was puriﬁed over SiO₂(gradient, 20−50% EtOAc in hexane)

1

to obtain methanesulfonamide 23 (0.67 g, 54%) as a white solid. H

NMR (300 MHz, CDCl₃) δ 8.25−8.19 (m, 2H), 7.80 (s, 1 H), 7.78−

7.70 (m, 3 H), 7.35 (d, J = 8.5 Hz, 2 H), 6.42 (br s, 1 H), 3.99 (s, 3

H), 3.09 (s, 3 H), 1.50 (s, 9 H).

N-[4-(2-tert-Butyl-4-chloroquinazolin-7-yl)phenyl]methane-

sulfonamide (31). A microwave vial was charged with quinazolinone

30 (500 mg, 1.78 mmol), 4-(methanesulfonamidophenyl)boronic acid

(421 mg, 1.96 mmol), Pd(PPh₃)₄(206 mg, 0.178 mmol), Na₂CO₃

(566 mg, 5.34 mmol), MeOH (3 mL), and toluene (1.5 mL). The

mixture was purged with argon for 5 min, and the vial was sealed. The

mixture was heated in a microwave reactor at 115 °C for 30 min. The

mixture was concentrated in vacuum and the residue triturated with

Et₂O to give the crude quinazoline as a brown solid. To a suspension

of the crude quinazolinone (445 mg, 1.2 mmol) and DIPEA (358 mg,

0.38 mL, 1.83 mmol) in benzene (3 mL) was added POCl₃(88 μL,

0.97 mmol), and the mixture was heated at reﬂux for 6.5 h. The

reaction mixture was cooled to room temperature and concentrated to

dryness. The residue was diluted with EtOAc, washed with water, 1 N

HCl, water, brine, dried over MgSO₄, and concentrated to give the

5-Bromo-7-tert-butyl-8-methoxy-2-(4-nitrophenyl)quinoline

(26). A mixture of 5-bromo-3-tert-butyl-2-methoxyaniline 25 (2.0 g,

7.75 mmol) and 4-nitrobenzaldehyde (1.4 g, 11.62 mmol) in CH₃CN

(10 mL) was stirred at room temperature for 45 min. BiCl₃(0.49 g,

1.55 mmol) was added to this mixture followed by a solution of N-

vinylpyrrolidin-2-one (1.72 g, 15.5 mmol) in CH₃CN (10 mL)

dropwise. The resulting mixture was stirred for 16 h and then diluted

with EtOAc and water (20 mL). A white solid precipitated and was

ﬁltered. The organic layer in the ﬁltrate was dried over Na₂SO₄,

ﬁltered, and concentrated. The crude product was puriﬁed over SiO₂

(gradient, 15−75% EtOAc in hexane) to aﬀord a mixture of

tetrahydroquinolines (978 mg) and compound 26 (312 mg).

To a solution of the mixture of tetrahydroquinolines (705 mg, 1.4

mmol) in CH₃CN (10 mL) at 0 °C was added dropwise a solution of

CAN (1.92 g, 3.51 mmol) in CH₃CN (10 mL). The reaction mixture

was stirred at 0 °C until all of the starting material was consumed. The

mixture was diluted with EtOAc and water. The organic layer was

separated, washed with brine, dried over MgSO₄, ﬁltered, and

concentrated. The crude product was puriﬁed over SiO₂(gradient,

1−15% EtOAc in hexane) to aﬀord quinoline 26 (302 mg, 622 mg

1

crude chloroquinazoline 31 (406 mg, 87%) as an orange solid. H

NMR (300 MHz, CDCl₃) δ 8.25 (d, J = 8.3 Hz, 1 H), 8.18 (d, J = 1.3

Hz, 1 H), 7.85 (dd, J = 8.8, 1.3 Hz, 1 H), 7.76 (d, J = 8.5 Hz, 2 H),

7.39 (d, J = 8.5 Hz, 2 H), 7.12 (br s, 1 H), 3.11 (s, 3 H), 1.51 (s, 9 H).

LC/MS (ES/APCI): 388.0 [M − H]⁻.

N-{4-[6-tert-Butyl-8-(5-ﬂuoro-2-oxo-1,2-dihydropyridin-3-

yl)-5-methoxyquinolin-3-yl]phenyl}methanesulfonamide (33)

Hydrobromide. A mixture of the 8-bromoquinoline 14 (150 mg,

0.324 mmol), (5-ﬂuoro-2-methoxy-3-pyridyl)boronic acid (112 mg,

0.657 mmol), Pd(PPh₃)₄(40 mg, 0.035 mmol), and Na₂CO₃(179 mg,

1.68 mmol) was suspended in MeOH (1.6 mL) and CH₂Cl₂(0.4 mL).

The reaction mixture was heated in a microwave reactor at 115 °C for

1 h. After cooling the mixture to room temperature, it was poured into

saturated aqueous NaHCO₃and extracted with EtOAc. The organic

layer was separated and washed with saturated aqueous NaHCO₃,

brine, dried over Na₂SO₄, ﬁltered, and concentrated under vacuum.

The product was puriﬁed over SiO₂(gradient, 27−50% EtOAc in

hexane) to yield N-[4-[6-tert-butyl-8-(5-ﬂuoro-2-methoxy-3-pyridyl)-

5-methoxy-3-quinolyl]phenyl]methanesulfonamide (121 mg, 73%) as

a white solid. ¹H NMR (300 MHz, CDCl₃) δ 9.05 (d, J = 2.3 Hz, 1 H),

8.53 (d, J = 2.3 Hz, 1 H), 8.08 (d, J = 3.0 Hz, 1 H), 7.78 (s, 1H), 7.72

(d, J = 8.7 Hz, 2 H), 7.75 (dd, J = 8.3, 3.0 Hz, 1 H), 7.38 (d, J = 8.3

Hz, 2 H), 6.60 (s, 1 H), 4.05 (s, 3 H), 3.89 (s, 3 H), 3.1 (s, 3 H), 1.54

(s, 9 H).

1

total, 22% overall yield) as a light yellow solid. H NMR (300 MHz,

CDCl₃) δ 8.58 (d, J = 8.9 Hz, 1 H), 8.46−8.35 (m, 4 H), 8.0 (d, J = 8.9

Hz, 1 H), 7.88 (s, 1 H), 4.36 (s, 3 H), 1.56 (s, 9 H). LC/MS (ES/

APCI): 416.0 [M + H]⁺.

N-[4-(5-Bromo-7-tert-butyl-8-methoxy-2-quinolyl)phenyl]-

methanesulfonamide (27). A solution of quinoline 26 (300 mg,

0.72 mmol), iron powder (121 mg, 2.17 mmol), and NH₄Cl (116 mg,

2.17 mmol) in a mixture of MeOH (30 mL) and water (10 mL) was

heated at reﬂux for 1.5 h. The reaction mixture was cooled to room

temperature and ﬁltered through a pad of Celite. The ﬁltrate was

concentrated, and the residue was partitioned between EtOAc and

water. The organic layer was dried over MgSO₄, ﬁltered, and

concentrated to aﬀord 4-(5-bromo-7-tert-butyl-8-methoxy-2-quinolyl)-

aniline (204 mg, 73%) which was used without further puriﬁcation. ¹H

NMR (400 MHz, CDCl₃) δ 8.40 (d, J = 8.8 Hz, 1 H), 8.11 (d, J = 8.4

Hz, 2 H), 7.86 (d, J = 8.8 Hz, 1 H), 7.73 (s, 1 H), 6.81 (d, J = 8.4 Hz, 2

H), 4.34 (s, 3 H), 3.93 (br s, 1 H), 1.55 (s, 9 H).

To a solution of 4-(5-bromo-7-tert-butyl-8-methoxy-2-quinolyl)-

aniline (200 mg, 0.52 mmol) in CH₂Cl₂(5 mL) at 0 °C was added

pyridine (92 μL, 1.14 mmol) followed by MsCl (42 μL, 0.55 mmol).

The reaction mixture was stirred at 0 °C for 45 min before it was

quenched with water and diluted with EtOAc. The organic layer was

separated, washed with brine, dried over MgSO₄, ﬁltered, and

concentrated. The crude was puriﬁed over SiO₂(gradient, 20−70%

EtOAc in hexane) to aﬀord methanesulfonamide 27 (208 mg, 86%).

¹H NMR (400 MHz, CDCl₃) δ 8.50 (d, J = 9.2 Hz, 1 H), 8.26 (d, J =

To a solution of N-[4-[6-tert-butyl-8-(5-ﬂuoro-2-methoxy-3-pyr-

idyl)-5-methoxy-3-quinolyl]phenyl]methanesulfonamide (110 mg,

0.26 mmol) in acetic acid (2.5 mL) was added an aqueous solution

of 48% HBr (0.06 mL, 1.1 mmol). The reaction vessel was sealed and

heated at 60 °C for 18 h. The mixture was cooled to room

temperature, and a yellow powder precipitated. The solid was ﬁltered,

washed with Et₂O, and dried in the oven under vacuum to give

1

pyridone 33 (71 mg, 55%) as a yellow solid. H NMR (400 MHz,

DMSO-d₆) δ 10.0 (s, 1 H), 9.19 (d, J = 2.0 Hz, 1 H), 8.67 (d, J = 2.0

Hz, 1 H), 7.93 (d, J = 8.1 Hz, 2 H), 7.84 (s, 1 H), 7.77 (dd, J = 8.1, 3.0

Hz, 1 H), 7.71 (br t, J = 3.1 Hz, 1 H), 7.40 (d, J = 8.6 Hz, 2 H), 4.03

(s, 3 H), 3.07 (s, 3 H), 1.50 (s, 9 H). LC/MS (ES): 496.07 (M + H)⁺.

N-[4-[6-tert-Butyl-8-(2,4-dioxopyrimidin-1-yl)-5-methoxy-3-

quinolyl]phenyl]methanesulfonamide (35). The introduction of

the uracil group was done following the procedure reported by

Wagner et al.²⁷A microwave vial was charged with 8-bromoquinoline

14 (200 mg, 0.431 mmol), uracil (72 mg, 0.642 mmol), N-(2-

cyanophenyl)pyridine-2-carboxamide (19 mg, 0.085 mmol), CuI (8

8.5 Hz, 2 H), 7.91 (d, J = 9.1 Hz, 1 H), 7.81 (s, 1 H), 7.37 (d, J = 8.8

Hz, 2 H), 6.53 (br s, 1 H), 4.35 (s, 3 H), 3.09 (s, 3 H), 1.51 (s, 9 H).

7-Bromo-2-tert-butyl-3H-quinazolin-4-one (30). To a solution

of 2-amino-4-bromobenzamide 29, (3.0 g, 14.0 mmol) and Et₃N (1.84

g, 18.2 mmol) in CH₂Cl₂(40 mL) at 0 °C was added dropwise

pivaloyl chloride (1.68 g, 14.0 mmol). The resulting solution was

allowed to reach room temperature and stirred for 18 h. Another

portion of pivaloyl chloride (0.44 g) was added to the mixture at 0 °C,

K

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mg, 0.042 mmol), K₃PO₄(183 mg, 0.86 mmol), CH₂Cl₂, and MeOH.

The suspension was degassed with argon followed by the addition of

DMSO (1.5 mL). The vial was sealed and irradiated in a microwave

reactor at 150 °C for 5 h. The reaction mixture was cooled to room

temperature and diluted with EtOAc. The organic layer was washed

with 1 N NaHSO₄, dried over MgSO₄, ﬁltered, and concentrated. The

crude residue was puriﬁed over SiO₂(EtOAc) to aﬀord uracil 35 (17

DMSO (5 mL). The mixture was irradiated in a microwave reactor at

150 °C for 5 h. The reaction mixture was cooled to room temperature

and the pH adjusted to ∼2 with 2 N HCl. The mixture was extracted

with EtOAc, and the organic extract was washed sequentially with

water, brine, dried over MgSO₄, and concentrated. The crude residue

was puriﬁed over SiO₂(5/95, MeOH/CH₂Cl₂) to aﬀord quinoline 37

1

(9.3 mg, 14%). H NMR (300 MHz, DMSO-d₆) δ 11.49 (br s, 1 H),

1

mg, 8%) as a semisolid. H NMR (400 MHz, CDCl₃) δ 9.31 (br s, 1

10.09 (br s, 1 H), 8.29 (d, J = 8.8 Hz, 2 H), 8.12 (s, 2 H), 7.72 (d, J =

7.8 Hz, 1 H), 7.58 (s, 1 H), 7.38 (d, J = 8.9 Hz, 2 H), 5.71 (d, J = 7.8

Hz, 1 H), 4.33 (s, 3 H), 3.08 (s, 3 H), 1.47 (s, 9 H). LC/MS (ES/

APCI): 495.0 [M + H]⁺.

H), 8.97 (d, J = 1.7 Hz, 1 H), 8.35 (d, J = 1.7 Hz, 1 H), 7.84 (s, 1 H),

7.63 (br s, 1 H), 7.44 (d, J = 8.7 Hz, 2 H), 7.38 (d, J = 7.9 Hz, 1 H),

7.09 (d, J = 8.4 Hz, 2 H), 5.92 (d, J = 7.9 Hz, 1 H), 4.01 (s, 3 H), 2.95

(s, 3 H), 1.55 (s, 9 H). HRMS (ESI): calcd for C₂₅H₂₇O₅N₄S, 495.169

67; found, 495.167 63 [M + H]⁺.

N-[4-[2-tert-Butyl-4-(2,4-dioxopyrimidin-1-yl)quinazolin-7-

yl]phenyl]methanesulfonamide (38). A mixture of chloroquinazo-

line 31 (100 mg, 0.256 mmol), uracil (88 mg, 0.78 mmol), and

Cs₂CO₃(168 mg, 0.516 mmol) in DMSO (2 mL) was heated in a

sealed vial at 125 °C for 2.5 h. The crude mixture was diluted with

EtOAc and washed with water, brine, dried over MgSO₄, and

concentrated. The crude product was puriﬁed over SiO₂(gradient,

30−50% EtOAc in hexane) to give the desired product quinazoline 38

N-[4-[7-tert-Butyl-5-(2,4-dioxopyrimidin-1-yl)-8-methoxy-2-

naphthyl]phenyl]methanesulfonamide (36). A ﬂask was charged

with N-[4-(5-bromo-7-tert-butyl-8-methoxynaphthyl)phenyl]methane-

sulfonamide 23 (350 mg, 0.76 mmol), tert-butyl carbamate (124 mg,

1.06 mmol), Pd₂(dba)₃.CHCl₃(104 mg, 0.1 mmol), 2-di-tert-

butylphosphino-2′,4′,6′-triisopropylbiphenyl (145 mg, 0.34 mmol),

sodium tert-butoxide (107 mg, 1.11 mmol), and toluene (10 mL) to

give a white suspension. The mixture was ﬂushed with argon for 10

min and stirred at room temperature over 3 days. The mixture was

diluted with EtOAc and water and was neutralized with 1 N HCl. The

organic layer was separated, washed with brine, dried over MgSO₄,

ﬁltered, and concentrated. The crude product was puriﬁed over SiO₂

(gradient, 20−60% EtOAc in hexane) to give tert-butyl N-[3-tert-butyl-

6-[4-(methanesulfonamido)phenyl]-4-methoxy-1-naphthyl]carbamate

(196 mg) and used in the next reaction.

1

(54 mg, 45%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

11.75 (br s, 1 H), 10.06 (br s, 1 H), 8.28 (s, 1 H), 8.11−8.01 (m, 2 H),

8.01−7.91 (m, 3 H), 7.37 (d, J = 8.6 Hz, 2 H), 5.92 (d, J = 7.9 Hz, 1

H), 3.08 (s, 3 H), 1.47 (s, 9 H). LC/MS (ES/APCI): 464.0 [M −

H]⁻.

N-[4-[6-tert-Butyl-5-methoxy-8-(2-oxo-1H-pyridin-3-yl)-3-

quinolyl]phenyl]methanesulfonamide (39). A microwave vial was

charged with 3-bromoquinoline 17 (150 mg, 0.387 mmol), 4-

(methanesulfonamido)phenylboronic acid (125 mg, 0.581 mmol),

Pd(PPh₃)₄(45 mg, 0.038 mmol), Na₂CO₃(123 mg, 1.16 mmol),

MeOH (1.5 mL), and CH₂Cl₂(0.2 mL). The vial was sealed and

irradiated in a microwave reactor at 115 °C for 1 h. The reaction

mixture was cooled to room temperature and diluted with EtOAc. The

organic layer was washed with water, dried over MgSO₄, ﬁltered, and

concentrated. The crude residue was puriﬁed over SiO₂(5/95,

MeOH/CH₂Cl₂) to aﬀord pyridone 39 (40 mg, 21%) as an oﬀ-white

A solution of tert-butyl N-[3-tert-butyl-6-[4-(methanesulfonamido)-

phenyl]-4-methoxy-1-naphthyl]carbamate (0.196 g, 0.2 mmol) and 4

M HCl in dioxane (0.49 mL, 1.97 mmol) in CH₂Cl₂(1.5 mL) was

stirred at room temperature for 3 h. The mixture was diluted with

EtOAc, poured over ice, neutralized with saturated aqueous NaHCO₃,

washed with brine, dried over MgSO₄, and concentrated. The crude

product was puriﬁed over SiO₂(gradient, 20−50% EtOAc in hexane)

to yield N-[4-(5-amino-7-tert-butyl-8-methoxy-2-naphthyl)phenyl]-

1

solid. H NMR (400 MHz, CDCl₃) δ 8.83 (d, J = 1.7 Hz, 1 H), 8.44

1

methanesulfonamide (73 mg) as a light brown solid. H NMR (300

(d, J = 1.7 Hz, 1 H), 7.73 (s, 1 H), 7.67 (dd, J = 6.7, 1.7 Hz, 1 H),

7.50−7.42 (m, 3 H), 7.20 (d, J = 8.8 Hz, 2 H), 6.49 (t, J = 6.7 Hz, 1

H), 4.01 (s, 3 H), 3.0 (s, 3 H), 1.54 (s, 9 H). HRMS (ESI): calcd for

C₂₆H₂₈O₄N₃S, 478.179 50; found, 478.177 70 [M + H]⁺.

N-[4-[6-tert-Butyl-5-methoxy-8-(6-methoxy-2-oxo-1H-pyri-

din-3-yl)-3-quinolyl]phenyl]methanesulfonamide (41) Hydro-

bromide. Using the two-step procedure for the preparation of 33

from 14 but using (2,6-dimethoxy-3-pyridyl)boronic acid, pyridone 41

(56 mg, 42% two steps) was obtained as a yellow solid. ¹H NMR (400

MHz, DMSO-d₆) δ 10.01 (br s, 1 H), 9.18 (d, J = 1.7 Hz, 1 H), 8.79

(br s, 1 H), 7.94 (d, J = 8.3 Hz, 2 H), 7.76 (s, 1 H), 7.67 (d, J = 8.1 Hz,

1 H), 7.40 (d, J = 8.3 Hz, 2 H), 6.29 (d, J = 7.5 Hz, 1 H), 4.04 (s, 3 H),

3.89 (s, 3 H), 3.07 (s, 3 H), 1.50 (s, 9 H). HRMS (ESI): calcd for

C₂₇H₃₀O₅N₃S, 508.190 07; found, 508.188 48 [M + H]⁺.

N-[4-[6-tert-Butyl-8-(2,4-dioxohexahydropyrimidin-1-yl)-5-

methoxy-3-quinolyl]phenyl]methanesulfonamide (42). Acrylic

acid (0.09 mL, 1.3 mmol) was added in three portions (0.02, 0.02, 0.05

mL) to a solution of N-[4-(8-amino-6-tert-butyl-5-methoxy-3-

quinolyl)phenyl]methanesulfonamide (prepared with the same

procedure as reported for N-[4-(5-amino-7-tert-butyl-8-methoxy-2-

naphthyl)phenyl]methanesulfonamide in experimental procedure for

36) (252 mg, 0.631 mmol) in toluene (2.5 mL) at room temperature.

After the ﬁrst addition, the reaction mixture was stirred at 120 °C for 2

h. After the second addition, the reaction mixture was stirred at 120 °C

for 1 h, and after the third addition, the reaction mixture was stirred at

120 °C overnight. The reaction mixture was cooled to room

temperature and concentrated. The residue was taken in glacial

AcOH (2 mL), and urea (95 mg, 1.58 mmol) was added. The reaction

mixture was stirred at 120 °C for 6 h and then cooled to room

temperature and the solvent evaporated. The dark brown residue was

partitioned between EtOAc and saturated aqueous NaHCO₃. The

aqueous layer was back-extracted twice with EtOAc. The combined

organic layers were dried over Na₂SO₄, ﬁltered, and evaporated. The

residue was puriﬁed over SiO₂(gradient, 50−80% EtOAc in CH₂Cl₂)

MHz, CDCl₃) δ 8.21 (br s, 1 H), 7.85 (d, J = 8.6 Hz, 1 H), 7.73 (d, J =

8.6 Hz, 2 H), 7.63 (d, J = 9.2 Hz, 1 H), 7.35 (d, J = 8.0 Hz, 2 H), 6.84

(br s, 1 H), 6.74 (br s, 1 H), 3.93 (s, 3 H), 3.07 (s, 3 H), 1.49 (s, 9 H).

In a ﬂask covered with foil, silver cyanate (135 mg, 0.9 mmol) was

heated under high vacuum at 50 °C for 18 h. To this dried material

was added (E)-3-methoxyacryloyl chloride (65 mg, 0.54 mmol) and

dry toluene (1.3 mL), and the slurry was heated at 120 °C under N₂

for 30 min. The mixture was cooled to room temperature, and the

precipitate was allowed to settle in an ice bath. In a separate dry ﬂask,

N-[4-(5-amino-7-tert-butyl-8-methoxy-2-naphthyl)phenyl]-

methanesulfonamide (0.072 g, 0.18 mmol) was taken up in DMF (1

mL) and cooled to 0 °C. To the DMF solution was added dropwise

the supernatant from the silver cyanate ﬂask over 10 min. The light

brown heterogeneous mixture formed was allowed to stir in an ice

bath for 30 min. The mixture was diluted with EtOAc, washed with

water, brine, and concentrated. The crude product was taken up in

EtOH (1 mL), and an 11% H₂SO₄solution in water (1 mL) was

added. The mixture was heated at 120 °C for 1.5 h until all solid was in

solution. The mixture was cooled to room temperature and poured

over ice, diluted with EtOAc, washed with water, brine, dried over

MgSO₄, ﬁltered, and concentrated. The crude product was puriﬁed

over SiO₂(gradient, 50−100% EtOAc in hexane) to give naphthalene

36 (60 mg, 67%) as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ

11.53 (br s, 1 H), 9.94 (br s, 1 H), 8.27 (br s, 1 H), 7.87 (br d, J = 8.7

Hz, 1 H), 7.82 (d, J = 8.7 Hz, 2 H), 7.72 (d, J = 7.9 Hz, 1 H), 7.67 (d, J

= 8.7 Hz, 1 H), 7.60 (s, 1 H), 7.37 (d, J = 8.6 Hz, 2 H), 5.72 (d, J = 7.7

Hz, 1 H), 4.02 (s, 3 H), 3.06 (s, 3 H), 1.48 (s, 9 H). LC/MS (ES/

APCI): 491.9 (M − H)⁻.

N-[4-[7-tert-Butyl-5-(2,4-dioxopyrimidin-1-yl)-8-methoxy-2-

quinolyl]phenyl]methanesulfonamide (37). A microwave vial was

charged with bromoquinoline 27 (60 mg, 0.13 mmol), uracil (73 mg,

0.65 mmol), N-(2-cyanophenyl)pyridine-2-carboxamide (14 mg, 0.063

mmol), CuI (6 mg, 0.03 mmol), K₃PO₄(137 mg, 0.64 mmol), and

L

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to give impure 42 (70 mg) as a greenish gray powder. The powder was

taken into a minimal amount of CH₂Cl₂and the insoluble material was

ﬁltered and rinsed with a small amount of CH₂Cl₂to give pure

dihydrouracil 42 (40 mg, 13%) as a light gray powder. ¹H NMR (300

MHz, DMSO-d₆) δ 10.33 (br s, 1 H), 9.20 (d, J = 1.9 Hz, 1 H), 8.53

(d, J = 2.0 Hz, 1 H), 7.84 (d, J = 8.6 Hz, 2 H), 7.70 (s, 1 H), 7.31 (d, J

= 8.5 Hz, 2 H), 4.00 (s, 3 H), 3.81 (br t, J = 6.5 Hz, 2 H), 2.98 (s, 3

H), 2.80 (t, J = 6.8 Hz, 2 H), 1.47 (s, 9 H). HRMS (ESI): calcd for

C₂₅H₂₉O₅N₄S, 497.185 32; found, 497.183 72 [M + H]⁺.

N-[[(3S)-1-[6-tert-Butyl-5-methoxy-8-(2-oxo-1H-pyridin-3-

yl)-3-quinolyl]pyrrolidin-3-yl]methyl]methanesulfonamide

(48). A screw-capped vial was charged with bromoquinoline 17 (301

mg, 0.77 mmol), N-(S)-1-pyrrolidin-3-ylmethylmethanesulfonamide

(200 mg, 0.779 mmol), Pd(OAc)₂(18 mg, 0.08 mmol), P(t-Bu)₃(24

μL, 0.079 mmol), t-BuONa (298 mg, 3.1 mmol), and toluene (3 mL).

The mixture was heated at 100 °C for 18 h. The reaction mixture was

cooled to room temperature and diluted with EtOAc. The organic

layer was washed with saturated aqueous NaHCO₃, dried over MgSO₄,

ﬁltered, and concentrated. The crude residue was puriﬁed over SiO₂

(gradient, 0−10% MeOH in CH₂Cl₂) to obtain product 48 (60 mg,

15%) as a solid. ¹H NMR (400 MHz, CDCl₃) δ 8.38 (d, J = 2.4 Hz, 1

H), 7.57 (dd, J = 6.8, 1.7 Hz, 1 H), 7.41 (s, 1 H), 7.36 (br d, J = 5.5

Hz, 1 H), 7.08 (d, J = 2.2 Hz, 1 H), 6.88 (br s, 1 H), 6.38 (t, J = 6.6

Hz, 1 H), 3.94 (s, 3 H), 3.47−3.34 (m, 2 H), 3.28 (q, J = 8.2 Hz, 1 H),

3.00−2.91 (m, 2 H), 2.89 (s, 3 H), 2.43 (quintuplet, J = 7.1 Hz, 1 H),

2.17−2.07 (m, 1 H), 1.89−1.77 (br s, 1 H), 1.68−1.58 (m, 1 H), 1.50

(s, 9 H). LC/MS (ES/APCI): 483.1 (M − H)⁻.

A solution of N-[[(3S)-1-(8-bromo-6-tert-butyl-5-methoxy-3-

quinolyl)pyrrolidin-3-yl]methyl]methanesulfonamide (6.13 g, 13

mmol), (2,6-dimethoxy-3-pyridyl)boronic acid (2.86 g, 15.6 mmol),

[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) chloride 1:1

complex with dichloromethane (412 mg, 0.5 mmol), and Cs₂CO₃

(12.76 g, 39.1 mmol in 13 mL of water) in dioxane (50 mL) was

purged with N₂for 10 min and stirred at 80 °C for 1 h. The reaction

was quenched with water (200 mL) and extracted with CH₂Cl₂. The

organic layer was washed with brine, dried over MgSO₄, and

concentrated. The crude product was puriﬁed over SiO₂(EtOAc) to

obtain N-[[(3S)-1-[6-tert-butyl-8-(2,6-dimethoxy-3-pyridyl)-5-me-

thoxy-3-quinolyl]pyrrolidin-3-yl]methyl]methanesulfonamide (6.26 g,

91%) as a yellow foam. ¹H NMR (300 MHz, DMSO-d₆) δ 8.42 (d, J =

2.7 Hz, 1 H), 7.58 (d, J = 7.9 Hz, 1 H), 7.25 (s, 1 H), 7.27−7.19 (m, 1

H), 7.06 (d, J = 2.7 Hz, 1 H), 6.44 (d, J = 7.9 Hz, 1 H), 3.93 (s, 3 H),

3.92 (s, 3 H), 3.76 (s, 3 H), 3.61−3.36 (m, 3 H), 3.21 (dd, J = 9.7, 6.6

Hz, 1 H), 3.06 (t, J = 6.4 Hz, 2 H), 2.93 (s, 3 H), 2.60−2.47 (m, 1 H),

2.23−2.09 (m, 1 H), 1.90−1.75 (m, 1 H), 1.45 (s, 9 H). LC/MS (ES/

APCI): 529.2 (M + H)⁺.

To a solution of N-[[(3S)-1-[6-tert-butyl-8-(2,6-dimethoxy-3-

pyridyl)-5-methoxy-3-quinolyl]pyrrolidin-3-yl]methyl]methane-

sulfonamide (120 mg, 0.227 mmol) in AcOH (2 mL) was added

aqueous 48% HBr solution (0.13 mL) at room temperature. The

mixture was stirred at 60 °C for 18 h. The reaction mixture was diluted

with EtOAc and washed with saturated aqueous NaHCO₃. The

aqueous layer was extracted with EtOAc, and the combined organic

layers were washed with saturated aqueous NaHCO₃, dried over

MgSO₄, and concentrated. The crude product was puriﬁed over SiO₂

(gradient, 0−5% MeOH in CH₂Cl₂) to give pyrrolidine 50 (47 mg,

40%) as a yellow foam. ¹H NMR (400 MHz, DMSO-d₆) δ 8.45 (d, J =

2.7 Hz, 1 H), 7.56 (d, J = 8.1 Hz, 1 H), 7.32 (s, 1 H), 7.23 (t, J = 5.9

Hz, 1 H), 7.09 (d, J = 2.6 Hz, 1 H), 6.26 (br s, 1 H), 3.93 (s, 3 H),

3.87 (s, 3 H), 3.63−3.50 (m, 2 H), 3.49−3.41 (m, 1 H), 3.23 (dd, J =

9.6, 6.3 Hz, 1 H), 3.08 (t, J = 6.3 Hz, 2 H), 2.94 (s, 3 H), 2.62−2.51

(m, 1 H), 2.13−2.12 (m, 1 H), 1.90−1.79 (m, 1 H), 1.46 (s, 9 H).

HRMS (ESI): calcd for C₂₆H₃₅O₅N₄S, 515.232 27; found, 515.230 59

[M + H]⁺.

N-[[(3S)-1-[6-tert-butyl-5-methoxy-8-(6-methoxy-2-oxo-1H-

pyridin-3-yl)-3-quinolyl]pyrrolidin-3-yl]methyl]methane-

sulfonamide (50). A mixture of 3,8-dibromoquinoline 13 (1.876 g,

5.03 mmol), tert-butyl N-[[(3S)-pyrrolidin-3-yl]methyl]carbamate

(1.52 g, 7.6 mmol), sodium tert-butoxide (728 mg, 7.49 mmol),

(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (582 mg,

1.01 mmol), and Pd₂(dba)₃(460 mg, 0.5 mmol) in degassed toluene

(10 mL) was warmed slowly to 100 °C and kept at this temperature

for 22 h. The reaction mixture was cooled to room temperature and

partitioned between EtOAc and saturated aqueous NH₄Cl. The

aqueous layer was extracted with EtOAc. The combined organic layers

were dried over MgSO₄and concentrated. The crude product was

puriﬁed over SiO₂(gradient, 10−50% EtOAc in hexane) to aﬀord tert-

butyl N-[[(3R)-1-(8-bromo-6-tert-butyl-5-methoxy-3-quinolyl)-

pyrrolidin-3-yl]methyl]carbamate (1.77 g, 71%) as a light green

N-[4-[8-(2,4-Dioxopyrimidin-1-yl)-5-methoxy-6-(triﬂuoro-

methyl)-3-quinolyl]phenyl]methanesulfonamide (51). A mix-

ture of Cu(I) (10.03 g, 52.6 mmol) and CsF (21.40 g, 140.8 mmol)

was ﬁnely ground in a mortar while in a glovebag under nitrogen

atmosphere to aﬀord a free-ﬂowing powder and transferred to a ﬂask.

The ﬂask was then charged with 2-iodo-5-nitroanisole (15.17 g, 54.3

mmol) and sulfolane (30 mL), and the mixture was stirred rapidly at

45 °C. To this mixture was added trimethyl(triﬂuoromethyl)silane

(19.2 g, 20 mL, 135 mmol) dropwise over 4 h using a syringe pump,

and the resulting mixture was stirred at room temperature for 18 h.

The mixture was diluted with EtOAc (500 mL) and stirred with Celite

for 5 min. The reaction mixture was ﬁltered through a pad of Celite,

diluted with EtOAc (1 L), and washed with 10% NH₄OH, 1.0 N HCl,

brine, dried over MgSO₄, ﬁltered, and concentrated. The amber

residue was puriﬁed over SiO₂(gradient, 0−40% CH₂Cl₂in hexane) to

aﬀord 2-methoxy-4-nitro-1-(triﬂuoromethyl)benzene (8.61 g, 72%) as

1

foam. H NMR (400 MHz, CDCl₃) δ 8.53 (d, J = 2.8 Hz, 1 H), 7.66

(s, 1 H), 7.07 (d, J = 2.5 Hz, 1 H), 4.66 (br s, 1 H), 3.85 (s, 3 H),

3.56−3.48 (m, 2 H), 3.46−3.38 (m, 1 H), 3.26−3.11 (m, 3 H), 2.63−

2.50 (m, 1 H), 2.21−2.10 (m, 1 H), 1.86−1.74 (m, 1 H), 1.40 (s, 9 H),

1.39 (s, 9 H). LC/MS (ES): 492 (M + H)⁺.

A solution of tert-butyl N-[[(3R)-1-(8-bromo-6-tert-butyl-5-me-

thoxy-3-quinolyl)pyrrolidin-3-yl]methyl]carbamate (1.76 g, 3.6

mmol) and 4 M HCl in dioxane (9 mL, 36 mmol) in CH₂Cl₂(11

mL) was stirred at room temperature for 16 h. The mixture was

concentrated, and the residual orange oil was treated with dry Et₂O

(150 mL) and stirred at room temperature for 30 min. The

supernatant was removed carefully and residual solid was dried

under high vacuum to give [(3R)-1-(8-bromo-6-tert-butyl-5-methoxy-

3-quinolyl)pyrrolidin-3-yl]methanamine (1.7 g) as an orange solid.

This product was used in the next reaction without puriﬁcation. To a

solution of the primary amine (1.7 g, 3.4 mmol) and Et₃N (2.36 mL,

16.9 mmol) in CH₂Cl₂(34 mL) was added methanesulfonyl chloride

(0.29 mL, 3.8 mmol) at 0 °C. The reaction mixture was stirred at

room temperature for 1 h. The reaction was quenched with water, and

the aqueous portion was extracted with EtOAc. The organic layer was

dried over MgSO₄and concentrated in vacuo to give N-[[(3S)-1-(8-

bromo-6-tert-butyl-5-methoxy-3-quinolyl)pyrrolidin-3-yl]methyl]-

1

a yellow crystalline solid. H NMR (300 MHz, CDCl₃) δ 7.95−7.83

(m, 2 H), 7.77 (d, J = 8.2 Hz, 1 H), 4.03 (s, 3 H). LC/MS (ES/APCI):

220.9 (M − H)⁻.

A Parr hydrogenation ﬂask was charged with 2-methoxy-4-nitro-1-

(triﬂuoromethyl)benzene (8.60 g, 38.9 mmol), 10% Pd/C (1.75 g),

and EtOH (150 mL). The ﬂask was placed under 57 psi of hydrogen

pressure at 55 °C for 18 h. The reaction mixture was cooled and the

catalyst ﬁltered and washed with isopropanol. The solvent was

removed and the crude product was puriﬁed over SiO₂(gradient, 0−

40% EtOAc in hexane) to aﬀord 3-methoxy-4-(triﬂuoromethyl)aniline

(7.18 g, 96%) as a waxy oﬀ-white solid. ¹H NMR (300 MHz, DMSO-

d₆) δ 7.17 (d, J = 8.2 Hz, 1 H), 6.30 (br s, 1 H), 6.16 (dd, J = 8.2, 1.3

Hz, 1 H), 5.77 (s, 2 H), 3.75 (s, 3 H). LC/MS (ES/APCI): 192.1 (M

+ H)⁺.

1

methanesulfonamide (1.44 g, 89%) as an oil. H NMR (400 MHz,

DMSO-d₆) δ 8.59 (d, J = 2.5 Hz, 1 H), 7.61 (s, 1 H), 7.23 (t, J = 6.2

Hz, 1 H), 7.04 (d, J = 2.7 Hz, 1 H), 3.90 (s, 3 H), 3.64−3.53 (m, 2 H),

3.52−3.43 (m, 1 H), 3.28−3.21 (m, 1 H), 3.07 (t, J = 6.8 Hz, 2 H),

2.93 (s, 3 H), 2.60−2.44 (m, 1 H), 2.24−2.12 (m, 1 H), 1.90−1.78

(m, 1 H), 1.44 (s, 9 H).

To a solution of 3-methoxy-4-(triﬂuoromethyl)aniline (3.01, 15.7

mmol), AcOH (7.5 mL), and anhydrous dioxane (25 mL) was added a

solution of Br₂(2.7 g, 16.9 mmol in 20 mL of dioxane) dropwise using

M

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Article

a syringe pump over 30 min at 0 °C. The reaction mixture was stirred

at room temperature for 1 h, poured into a mixture of 1.0 M NaOH

(150 mL) and 2.0 M Na₂CO₃(150 mL), and extracted with CH₂Cl₂.

The combined extracts were washed sequentially with 0.5 M Na₂CO₃,

brine, dried over Na₂SO₄, ﬁltered, and concentrated to aﬀord 2-

bromo-5-methoxy-4-(triﬂuoromethyl)aniline (4.10 g, 97%) as a black

crystalline solid. ¹H NMR (400 MHz, DMSO-d₆) δ 7.44 (s, 1 H), 6.57

(s, 1 H), 3.77 (s, 3 H).

To a solution of 2-bromoprop-2-enal (1.91 g) in AcOH (50 mL)

was added dropwise Br₂(0.72 mL, 13.9 mmol). To this solution was

added 2-bromo-5-methoxy-4-(triﬂuoromethyl)aniline (3.48 g, 12.9

mmol), and the mixture was stirred at 100 °C for 1 h. The mixture was

cooled to room temperature and poured into a stirred ice cold solution

of 2.0 M NaOH (550 mL). To the mixture was slowly added 2.0 M

Na₂CO₃[vigorous foaming] until the solution reached pH 8. The

resulting solution extracted with CH₂Cl₂, and the organic extracts were

washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated. The

residue was puriﬁed over SiO₂(gradient, 0−100% CH₂Cl₂in hexane)

and rechromatographed (gradient, 0−100% EtOAc in hexane) to

aﬀord 3,8-dibromo-5-methoxy-6-(triﬂuoromethyl)quinoline (938 mg,

19%) as an oﬀ-white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 9.26 (d,

J = 2.2 Hz, 1 H), 8.91 (d, J = 2.2 Hz, 1 H), 8.33 (s, 1 H), 4.06 (s, 3 H).

mixture was partitioned between EtOAc and water, and the organic

layer was dried over Na₂SO₄, ﬁltered, and concentrated. The residue

was taken into EtOH (1.5 mL) and water (1.5 mL), and concentrated

H₂SO₄(0.2 mL) was added. The resulting sticky slurry was stirred at

110 °C for 1 h. DMF was added and stirred at 110 °C for 2 h before

cooling to room temperature. The clear solution was partitioned

between EtOAc and saturated aqueous NaHCO₃. The organic layer

was dried over Na₂SO₄, ﬁltered, and concentrated. The residue was

puriﬁed over SiO₂(gradient, 0−40% EtOAc in CH₂Cl₂) to yield

triﬂuoromethylquinoline 51 (20 mg, 16%). ¹H NMR (400 MHz,

CDCl₃) δ 9.11 (d, J = 2.3 Hz, 1 H), 8.89 (br s, 1 H), 8.59 (br s, 1 H),

8.56 (d, J = 2.3 Hz, 1 H), 7.72 (d, J = 8.7 Hz, 2 H), 7.43 (d, J = 8.6 Hz,

2 H), 6.79 (br s, 1 H), 4.04 (s, 3 H), 3.11 (s, 3 H).

N-[4-[8-(2,4-Dioxopyrimidin-1-yl)-5-methoxy-6-(2,2,2-tri-

ﬂuoroethyl)-3-quinolyl]phenyl]methanesulfonamide (52). In a

ﬂask was dissolved 2-methoxy-4-nitrobenzaldehye (9.93 g, 54.8 mmol)

in anhydrous DME (100 mL), and the mixture was stirred to obtain a

clear yellow solution. To this solution was added sequentially

CF₃SiMe₃(9.0 mL, 8.66 g, 60.9 mmol) and CsF (792 mg, 5.2

mmol). The mixture was sonicated for 20 min and then stirred at

room temperature for 40 min. A 2.0 M HCl solution (100 mL) was

added and the resulting mixture stirred at room temperature for 1 h.

EtOAc was added to the mixture. The two layers were separated, and

the organic phase was washed with saturated aqueous NaHCO₃, brine,

dried over MgSO₄, ﬁltered, and concentrated. The crude product was

puriﬁed over SiO₂(gradient, 0−100% CH₂Cl₂in hexane) to give 2,2,2-

triﬂuoro-1-(2-methoxy-4-nitrophenyl)ethanol (13.1 g, 95%) as a white

A

vial was charged with 3,8-dibromo-5-methoxy-6-

(triﬂuoromethyl)quinoline (850 mg, 2.2 mmol), N-[4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methanesulfonamide (661

mg, 2.22 mmol), an aqueous solution of Cs₂CO₃(2.2 g, 6.7 mmol in

2.3 mL of water), and dioxane (10 mL), and the mixture was sparged

with N₂for 10 min. Pd(dppf)Cl₂·CH₂Cl₂(74 mg, 0.09 mmol) was

added to the mixture and stirred at 65 °C for 110 min. The mixture

was cooled and poured into CH₂Cl₂and 0.5 M Na₂CO₃. The phases

were separated and washed sequentially with water, brine, dried over

MgSO₄, ﬁltered, and concentrated. The product was puriﬁed over SiO₂

(gradient, 0−100% EtOAc in CH₂Cl₂) to aﬀord N-[4-[8-bromo-5-

methoxy-6-(triﬂuoromethyl)-3-quinolyl]phenyl]methanesulfonamide

(538 mg, 51%) as a light amber solid. ¹H NMR (300 MHz, DMSO-d₆)

δ 10.07 (br s, 1 H), 9.53 (d, J = 2.2 Hz, 1 H), 8.73 (d, J = 2.1 Hz, 1 H),

8.27 (s, 1 H), 8.0 (d, J = 8.6 Hz, 2 H), 7.42 (d, J = 8.6 Hz, 2 H), 4.11

(s, 3 H), 3.09 (s, 3 H).

1

solid. H NMR (300 MHz, DMSO-d₆) δ 7.93 (dd, J = 8.4, 2.0 Hz, 1

H), 7.84 (d, J = 2.0 Hz, 1 H), 7.77 (d, J = 8.6 Hz, 1 H), 7.08 (d, J = 5.8

Hz, 1 H), 5.49 (quintuplet, J = 6.5 Hz, 1 H), 3.97 (s, 3 H).

To a solution of 2,2,2-triﬂuoro-1-(2-methoxy-4-nitrophenyl)ethanol

(13.01 g, 51.8 mmol) in THF (300 mL) was added NaH (2.25 g, 56.2

mmol, 60 wt % dispersion in mineral oil) at room temperature. The

mixture was sonicated for 20 min, then stirred at room temperature for

10 min. A solution of p-toluenesulfonyl chloride (11.86 g, 62.2 mmol)

dissolved in THF (100 mL) was added at room temperature and the

mixture stirred at 50 °C for 1.5 h. The solution was cooled, poured

into 0.5 M NaHCO₃, and diluted with EtOAc. The organic phase was

separated, washed with brine, dried over MgSO₄, ﬁltered, and

concentrated. The crude product was puriﬁed over SiO₂(gradient,

0−100% CH₂Cl₂in hexane) to aﬀord [2,2,2-triﬂuoro-1-(2-methoxy-4-

nitrophenyl)ethyl] 4-methylbenzenesulfonate (20.36 g, 97%) as a

A vial was charged with N-[4-[8-bromo-5-methoxy-6-(triﬂuoro-

methyl)-3-quinolyl]phenyl]methanesulfonamide (370 mg, 0.78

mmol), tert-butyl carbamate (110 mg, 0.94 mmol), Pd₂(dba)₃·CHCl₃

(120 mg, 0.116 mmol), 2-di-tert-butylphosphino-2′,4′,6′-triisopropyl-

1′,1′-biphenyl (150 mg, 0.353 mmol), sodium tert-butoxide (110 mg,

1.14 mmol), and toluene (3.5 mL), and the resulting mixture was

degassed by bubbling argon for 10 min. The reaction mixture was

stirred at room temperature for 2 days and then diluted with EtOAc

and washed with saturated aqueous NH₄Cl. The organic layer was

dried over Na₂SO₄, ﬁltered, and concentrated. The residue was

puriﬁed over SiO₂(gradient, 0−10% EtOAc in CH₂Cl₂) to obtain tert-

butyl N-[3-[4-(methanesulfonamido)phenyl]-5-methoxy-6-(triﬂuoro-

methyl)-8-quinolyl]carbamate (398 mg, 78%) as a light brown foam.

A mixture of tert-butyl N-[3-[4-(methanesulfonamido)phenyl]-5-

methoxy-6-(triﬂuoromethyl)-8-quinolyl]carbamate (390 mg, 0.16

mmol), 4 M HCl in dioxane (4 mL), and CH₂Cl₂(4 mL) was stirred

at room temperature for 4 h and then concentrated. The residue was

partitioned between EtOAc and saturated aqueous NaHCO₃. The

organic layer was separated, dried over Na₂SO₄, ﬁltered, and

concentrated to give N-[4-[8-amino-5-methoxy-6-(triﬂuoromethyl)-3-

1

white solid. H NMR (300 MHz, DMSO-d₆) δ 7.81−7.73 (m, 2 H),

7.69 (d, J = 8.4 Hz, 2 H), 7.60 (d, J = 8.5 Hz, 1 H), 7.35 (d, J = 8.4 Hz,

2 H), 6.24 (q, J = 6.4 Hz, 1 H), 3.95 (s, 3 H), 2.33 (s, 3 H).

A Parr hydrogenation ﬂask was charged with [2,2,2-triﬂuoro-1-(2-

methoxy-4-nitrophenyl)ethyl] 4-methylbenzenesulfonate (20.35 g,

50.2 mmol) and dissolved in EtOH (250 mL). To the solution was

added 10% Pd/C (4.02 g), and hydrogenation was under 55 psi of

hydrogen at 50 °C for 1.5 h. The catalyst was ﬁltered through a plug of

glass wool and washed with hot EtOH. The ﬁltrate was concentrated

and the tosylate salt precipitated as a white solid. The residue was

partitioned between 1.0 M NaOH and Et₂O. The organic extract was

washed with brine, dried over MgSO₄, ﬁltered, and concentrated. The

residue was puriﬁed over SiO₂(CH₂Cl₂) to obtain 3-methoxy-4-(2,2,2-

1

triﬂuoroethyl)aniline (9.04 g, 88%) as an oﬀ-white solid. H NMR

(300 MHz, DMSO-d₆) δ 6.87 (d, J = 8.1 Hz, 1 H), 6.24 (d, J = 2.0 Hz,

1 H), 6.13 (dd, J = 8.0, 2.0 Hz, 1 H), 5.19 (br s, 2 H), 3.70 (s, 3 H),

3.32 (q, J = 11.7 Hz, 2 H). LC/MS (ES/APCI): 206.1 (M + H)⁺.

To a solution of 3-methoxy-4-(2,2,2-triﬂuoroethyl)aniline (8.16 g,

39.8 mmol), AcOH (23 mL), and dioxane (100 mL) was added a

solution of Br₂(2.26 mL, 44 mmol) in dioxane (45 mL) dropwise over

30 min at 5 °C. The solution was warmed to room temperature and

stirred for 1 h. The mixture was poured into 1.0 M NaOH and

extracted with CH₂Cl₂. The combined extracts were washed with

brine, dried over MgSO₄, ﬁltered, and concentrated to aﬀord crude 2-

bromo-5-methoxy-4-(2,2,2-triﬂuoroethyl)aniline (11.42 g) as a pale

olive solid. ¹H NMR (300 MHz, DMSO-d₆) δ 7.21 (br s, 1 H), 6.48 (s,

1 H), 5.41 (br s, 2 H), 3.71 (s, 3 H), 3.38 (q, J = 11.6 Hz, 2 H). LC/

MS (ES/APCI): 284.0 (M + H)⁺.

1

quinolyl]phenyl]methanesulfonamide (300 mg, 73%) as a solid. H

NMR (400 MHz, DMSO-d₆) δ 9.21 (d, J = 2.1 Hz, 1 H), 8.53 (d, J =

2.3 Hz, 1 H), 7.93 (d, J = 8.6 Hz, 2 H), 7.41 (d, J = 8.5 Hz, 2 H), 6.97

(s, 1 H), 6.17 (br s, 2 H), 3.93 (s, 3 H), 3.08 (s, 3H).

(E)-3-Methoxyacryloyl chloride (90 mg, 1.12 mmol) was added at

room temperature to a suspension of a previously dried silver cyanate

(180 mg, 1.2 mmol) in toluene (2 mL). The resulting slurry was

stirred at reﬂux for 30 min before being cooled to 0 °C. After the solid

had settled, the supernatant was added dropwise to a solution of N-[4-

[8-amino-5-methoxy-6-(triﬂuoromethyl)-3-quinolyl]phenyl]-

methanesulfonamide (100 mg, 0.243 mmol) in DMF (1.5 mL) at 0

°C, and the resulting light orange mixture was stirred for 30 min. The

N

dx.doi.org/10.1021/jm401329s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

To a solution of 2-bromoprop-2-enal (2.71 g, 20.1 mmol) in AcOH

(25 mL) was added Br₂(3.2 g, 1.0 mL, 20 mmol) at 0 °C. This

solution was poured into a stirred solution of 2-bromo-5-methoxy-4-

(2,2,2-triﬂuoroethyl)aniline (5.67 g, 19.9 mmol) and AcOH (25 mL)

and the resulting mixture stirred at 100 °C for 2 h. The solution was

cooled, diluted with water, and extracted with EtOAc. The extract was

washed with 2.0 M NaOH, brine, dried over MgSO₄, ﬁltered, and

concentrated. The crude product was puriﬁed over SiO₂(CH₂Cl₂) to

obtain 3,8-dibromo-5-methoxy-6-(2,2,2-triﬂuoroethyl)quinoline (2.69

g, 34%) as a light orange solid. ¹H NMR (300 MHz, DMSO-d₆) δ 9.13

(d, J = 2.3 Hz, 1 H), 8.76 (d, J = 2.3 Hz, 1 H), 8.19 (s, 1 H), 3.95 (s, 3

H), 3.90 (q, J = 11.2 Hz, 2 H). LC/MS (ES/APCI): 399.9 (M + H)⁺.

A vial was charged with 3,8-dibromo-5-methoxy-6-(2,2,2-

triﬂuoroethyl)quinoline (2.62 g, 6.5 mmol), N-[4-(4,4,5,5-tetrameth-

yl-1,3,2-dioxaborolan-2-yl)phenyl]methanesulfonamide (1.92 g, 6.5

mmol), an aqueous solution of Cs₂CO₃(6.46 g, 19.8 mmol, 6 mL

of water), and dioxane (25 mL), and the mixture was sparged with N₂

for 10 min. Pd(dppf)Cl₂·CH₂Cl₂(161 mg, 0.19 mmol) was added to

the mixture, and the mixture was stirred at 50 °C for 2 h. The mixture

was cooled and poured into CH₂Cl₂and 0.5 M Na₂CO₃. The phases

were separated, and the organic phase was washed sequentially with

water, brine, dried over MgSO₄, ﬁltered, and concentrated. The

product was puriﬁed over SiO₂(gradient, 0−60% EtOAc in CH₂Cl₂)

to aﬀord N-[4-[8-bromo-5-methoxy-6-(2,2,2-triﬂuoroethyl)-3-

and the residue puriﬁed over SiO₂(gradient, 0−70% EtOAc in

CH₂Cl₂) to obtain a mixture of geometric isomers used in the next

reaction (204 mg).

A suspension of the product from previous reaction (178 mg) and

H₂SO₄(0.5 mL) with EtOH (2 mL) and water (2 mL) was stirred at

110 °C for 2 h. The mixture was cooled to room temperature and

allowed to stand overnight. A yellow solid crystallized from the yellow

supernatant. The solid was collected by ﬁltration and washed with

EtOH/water (1/1). The solid was dissolved in hot EtOH (10 mL) and

water (10 mL) added. The resulting solution was allowed to cool to

room temperature, and ﬂuﬀy white crystals formed. The solid was

collected by ﬁltration, washed with 1:1 EtOH/water (1/1), and dried

under vacuum to obtain the triﬂuoroethylquinoline 52 (107 mg, 6%)

1

as white crystals. H NMR (300 MHz, DMSO-d₆) δ 11.51 (d, J = 1.8

Hz, 1 H), 10.03 (s, 1 H), 9.34 (d, J = 2.2 Hz, 1 H), 8.66 (d, J = 2.0 Hz,

1 H), 7.94 (d, J = 8.7 Hz, 2 H), 7.90 (s, 1 H), 7.72 (d, J = 7.9 Hz, 1 H),

7.40 (d, J = 8.7 Hz, 2 H), 5.71 (dd, J = 7.9, 1.9 Hz, 1 H), 4.04 (s, 3 H),

3.96 (d, J = 11.1 Hz, 1 H), 3.88 (d, J = 11.1 Hz, 1 H), 3.08 (s, 3 H).

LC/MS (ES/APCI): 521.0 (M + H)⁺.

N-[4-[6-[1-(Diﬂuoromethyl)cyclopropyl]-8-(2,4-dioxopyrimi-

din-1-yl)-5-methoxy-3-quinolyl]phenyl]methanesulfonamide

(53). Br₂(4.48 mL, 86.9 mmol) in AcOH (50 mL) was added to a

solution of 2-bromoprop-2-enal (11.7 g, 86.7 mmol) in AcOH (100

mL) at room temperature until the solution showed a faint Br₂color.

To this solution was added methyl 4-amino-5-bromo-2-methoxyben-

zoate (22.6 g, 86.9 mmol). The resulting solution was gradually heated

to 100 °C, and stirring continued for 15 min. The mixture was cooled

to room temperature and concentrated. The residue was neutralized

with saturated aqueous NaHCO₃, and a precipitate formed. The

resulting solid was ﬁltered and washed with water and Et₂O to aﬀord

methyl 3,8-dibromo-5-methoxyquinoline-6-carboxylate (11.04 g, 34%).

¹H NMR (300 MHz, CDCl₃) δ 9.08 (d, J = 2.3 Hz, 1 H), 8.76 (d, J =

1

quinolyl]phenyl]methanesulfonamide (368 mg, 11%) as a solid. H

NMR (300 MHz, DMSO-d₆) δ 10.04 (s, 1 H), 9.40 (d, J = 2.2 Hz, 1

H), 8.62 (d, J = 2.2 Hz, 1 H), 8.13 (s, 1 H), 7.96 (d, J = 8.6 Hz, 2 H),

7.41 (d, J = 8.6 Hz, 2 H), 3.99 (s, 3 H), 3.90 (q, J = 11.2 Hz, 2 H), 3.09

(s, 3 H). LC/MS (ES/APCI): 489.0 (M + H)⁺.

A ﬂask was charged with N-[4-[8-bromo-5-methoxy-6-(2,2,2-

triﬂuoroethyl)-3-quinolyl]phenyl]methanesulfonamide (1.0 g, 2.04

mmol), tert-butyl carbamate (290 mg, 2.48 mmol), Pd₂(dba)₃·CHCl₃

(106 mg, 0.1 mmol), 2-di-tert-butylphosphino-2′,4′6′-triisopropyl-

1′,1′-biphenyl (150 mg, 0.353 mmol), sodium tert-butoxide (287

mg, 3.0 mmol), and toluene (10 mL), and the resulting mixture was

degassed by bubbling argon for 10 min. The reaction mixture was

stirred at room temperature for 1.5 h and then diluted with EtOAc and

washed with saturated aqueous NH₄Cl. The organic layer was washed

with brine, dried over Na₂SO₄, ﬁltered, and concentrated. The residue

was puriﬁed over SiO₂(gradient, 0−25% EtOAc in CH₂Cl₂) to obtain

tert-butyl N-[3-[4-(methanesulfonamido)phenyl]-5-methoxy-6-(2,2,2-

triﬂuoroethyl)-8-quinolyl]carbamate (926 mg, 86%) as a foam. ¹H

NMR (300 MHz, DMSO-d₆) δ 10.02 (s, 1 H), 9.24 (d, J = 2.1 Hz, 1

H), 8.88 (s, 1 H), 8.58 (d, J = 2.1 Hz, 1 H), 8.24 (s, 1 H), 7.93 (d, J =

8.7 Hz, 2 H), 7.41 (d, J = 8.7 Hz, 2 H), 3.96−3.79 (m, 2 H), 3.93 (s, 3

H), 3.08 (s, 3 H) 1.54 (s, 9 H). LC/MS (ES/APCI): 526.1 (M + H)⁺.

A mixture of tert-butyl N-[3-[4-(methanesulfonamido)phenyl]-5-

methoxy-6-(2,2,2-triﬂuoroethyl)-8-quinolyl]carbamate (908 mg, 1.73

mmol), 4 M HCl in dioxane (8 mL), and CH₂Cl₂(4 mL) was stirred

at room temperature for 2 h and then concentrated. The mixture was

partitioned between EtOAc and saturated aqueous NaHCO₃. The

organic layer was separated, washed with brine, dried over Na₂SO₄,

ﬁltered, and concentrated. The residue was puriﬁed over SiO₂

(gradient, 0−50% EtOAc in CH₂Cl₂) to give N-[4-[8-amino-5-

methoxy-6-(2,2,2-triﬂuoroethyl)-3-quinolyl]phenyl]-

2.4 Hz, 1 H), 8.46 (s, 1 H), 4.08 (s, 3 H), 4.01 (s, 3 H).

To a solution of methyl 3,8-dibromo-5-methoxyquinoline-6-

carboxylate (11.04 g, 29.5 mmol) in CH₂Cl₂(550 mL) at 0 °C was

added dropwise DIBAL (64.4 mL, 64.4 mmol, 1 M in CH₂Cl₂). The

reaction was quenched with aqueous Rochelle salt, and the mixture

was partitioned between water and CH₂Cl₂. The organic layer was

washed with water, dried over Na₂SO₄, ﬁltered, and concentrated. The

crude product was puriﬁed over SiO₂(gradient, 0−10% MeOH in

CH₂Cl₂) to aﬀord (3,8-dibromo-5-methoxy-6-quinolyl)methanol (9.5

g, 93%). ¹H NMR (300 MHz, CDCl₃) δ 9.01 (d, J = 2.2 Hz, 1 H), 8.59

(d, J = 2.2 Hz, 1 H), 8.18 (s, 1 H), 4.93 (d, J = 5.7 Hz, 2 H), 3.98 (s, 3

H), 1.98 (t, J = 5.8 Hz, 1 H).

A solution of (3,8-dibromo-5-methoxy-6-quinolyl)methanol (7.82 g,

22.5 mmol), CBr₄(8.97 g, 27.0 mmol), PPh₃(7.09 g, 27.0 mmol), and

CH₂Cl₂(250 mL) was stirred at room temperature for 18 h.

Additional CBr₄(3.73, 11.2 mmol) and PPh₃(2.95 g, 11.2 mmol)

were added to the mixture and stirred for 1 h. The crude reaction

mixture was concentrated and the residue puriﬁed over SiO₂(gradient,

0−100% CH₂Cl₂in hexane) to aﬀord 3,8-dibromo-6-(bromomethyl)-

1

5-methoxyquinoline (7.62 g, 83%) as a white solid. H NMR (300

MHz, CDCl₃) δ 9.01 (d, J = 2.3 Hz, 1 H), 8.58 (d, J = 2.2 Hz, 1 H),

8.07 (s, 1 H), 4.67 (s, 2 H), 4.06 (s, 3 H).

A mixture of 3,8-dibromo-6-(bromomethyl)-5-methoxyquinoline

(8.0 g, 19.5 mmol), KCN (12.4 g, 191 mmol), CH₂Cl₂(156 mL), and

water (140 mL) was heated at reﬂux for 15 min. The reaction mixture

was diluted with water and extracted with CH₂Cl₂. The organic layer

was dried over Na₂SO₄and concentrated. The residue was puriﬁed

over SiO₂(gradient, 0−5% MeOH in CH₂Cl₂) to aﬀord 2-(3,8-

dibromo-5-methoxy-6-quinolyl)acetonitrile (4.53 g, 65%) as a white

1

methanesulfonamide (554 mg, 75%) as a yellow solid. H NMR (300

MHz, DMSO-d₆) δ 9.98 (s, 1 H), 9.08 (d, J = 2.2 Hz, 1 H), 8.42 (d, J

= 2.1 Hz, 1 H), 7.89 (d, J = 8.7 Hz, 2 H), 7.39 (d, J = 8.5 Hz, 2 H),

6.80 (s, 1 H), 5.87 (s, 2 H), 3.84 (s, 3 H), 3.72 (q, J = 11.4 Hz, 2 H),

3.07 (s, 3 H). LC/MS (ES/APCI): 426.0 (M + H)⁺.

A suspension of dried silver cyanate (529 mg, 3.5 mmol) and

anhydrous toluene (3 mL) was stirred at 90 °C for 20 min. A solution

of (E)-3-methoxyprop-2-enoyl chloride (216 mg, 1.8 mmol) in

anhydrous toluene (2 mL) was added dropwise to the suspension

and stirred at 90 °C for 30 min. The suspension was allowed to stand

at room temperature for 10 min, and the supernatant was transferred

by syringe to a solution of N-[4-[8-amino-5-methoxy-6-(2,2,2-

triﬂuoroethyl)-3-quinolyl]phenyl]methanesulfonamide (249 mg, 0.58

mmol) in DMF (3 mL) at −20 °C. The mixture was allowed to reach

room temperature and stirred for 18 h. The mixture was concentrated

1

solid. H NMR (300 MHz, CDCl₃) δ 9.04 (d, J = 2.2 Hz, 1 H), 8.57

(d, J = 2.3 Hz, 1 H), 8.09 (s, 1 H), 4.01 (s, 3 H), 3.93 (s, 2 H).

A mixture of 2-(3,8-dibromo-5-methoxy-6-quinolyl)acetonitrile

(5.02 g, 14.1 mmol), 1,2-dibromoethane (1.46 mL, 16.9 mmol), and

DMF (60 mL) was cooled to 0 °C, and NaH (1.69 g, 42.3 mmol, 60%

mineral oil dispersion) was added. The mixture was warmed to room

temperature and stirred for 1 h. The reaction mixture was diluted with

water and extracted with CH₂Cl₂. The organic layer was washed with

water, dried over Na₂SO₄, ﬁltered, and concentrated. The crude

O

dx.doi.org/10.1021/jm401329s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

product was puriﬁed over SiO₂(gradient, 0−100% CH₂Cl₂in hexane)

to aﬀord 1-(3,8-dibromo-5-methoxy-6-quinolyl)cyclopropane-

carbonitrile (1.91 g, 35%) as a solid. H NMR (400 MHz, CDCl₃)

δ 9.03 (d, J = 2.0 Hz, 1 H), 8.61 (d, J = 2.1 Hz, 1 H), 8.00 (s, 1 H),

4.18 (s, 3 H), 1.83 (dd, J = 7.9, 5.3 Hz, 2 H), 1.49 (dd, J = 7.5, 4.9 Hz,

2 H).

with CH₂Cl₂. The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated. The crude material was puriﬁed over SiO₂(gradient,

30−80% EtOAc in hexane) to aﬀord N-[4-[8-amino-6-[1-

(diﬂuoromethyl)cyclopropyl]-5-methoxy-3-quinolyl]phenyl]-

1

methanesulfonamide (110 mg, 90%) as a solid. H NMR (400 MHz,

CDCl₃) δ 8.98 (d, J = 2.2 Hz, 1 H), 8.43 (d, J = 2.2 Hz, 1 H), 7.72 (d,

J = 8.4 Hz, 2 H), 7.38 (d, J = 8.4 Hz, 2 H), 6.90 (s, 1 H), 6.49 (br s, 1

H), 6.05 (t, J = 58.4 Hz, 1 H), 4.83 (br s, 2 H), 3.98 (s, 3 H), 3.10 (s, 3

H), 1.31 (dd, J = 6.7, 5.0 Hz, 2 H), 1.14−1.08 (br m, 2 H).

In a ﬂask, (E)-3-methoxyacryloyl chloride (91.8 mg, 0.761 mmol)

and dried silver cyanate (190 mg, 1.27 mmol) were combined with

toluene (2 mL), and the mixture was heated to 130 °C and stirred for

30 min. The mixture was cooled to 0 °C and allowed to settle. The

supernatant was added dropwise to N-[4-[8-amino-6-[1-

(diﬂuoromethyl)cyclopropyl]-5-methoxy-3-quinolyl]phenyl]-

methanesulfonamide (110 mg, 0.25 mmol) in DMF (1 mL) at 0 °C.

The mixture was stirred at 0 °C for 45 min and then diluted with

EtOAc and washed with water and brine. The organic layer was dried

over Na₂SO₄, ﬁltered, and concentrated. The crude reaction mixture

was puriﬁed over SiO₂(gradient, 50−100% EtOAc in hexane) to

obtain a mixture of geometric isomers used in the next reaction (160

mg) as a light yellow solid.

A slurry of the product from previous reaction (160 mg) and

concentrated H₂SO₄(0.35 mL) with EtOH (4 mL) and water (3 mL)

was heated at 110 °C for 1.5 h. Most of EtOH was evaporated, and the

residue was neutralized with saturated aqueous NaHCO₃. The mixture

was extracted with EtOAc. The organic layer was dried over Na₂SO₄

and concentrated. The crude product was concentrated and triturated

with Et₂O and dried to yield the cyclopropylquinoline 53 (123 mg,

93%) as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.46 (d, J

= 1.5 Hz, 1 H), 10.0 (s, 1 H), 9.31 (d, J = 2.3 Hz, 1 H), 8.65 (d, J = 2.0

Hz, 1 H), 7.93 (d, J = 8.6 Hz, 2 H), 7.87 (s, 1 H), 7.69 (d, J = 8.0 Hz, 1

H), 7.39 (d, J = 8.6 Hz, 2 H), 6.15 (t, J = 57.0 Hz, 1 H), 5.69 (dd, J =

7.8, 1.8 Hz, 1 H), 4.12 (s, 3 H), 3.07 (s, 3 H), 1.32 (br s, 2 H), 1.21−

1.15 (m, 2 H). LC/MS (ES/APCI): 529.0 (M + H)⁺.

HCV NS5B Polymerase Biochemical Assay Protocol. The

enzymatic activity of HCV polymerase (NS5B570n-Con1) was

measured as described previously¹⁵with only two changes as follows:

concentration of RNA template was 3 nM and concentration of

NS5B570n-Con1 enzyme was 3 nM.

Stable HCV Replicon Assay. The stable GT-1a H77 and GT-1b

Con1 subgenomic replicon cell lines expressing the Renilla luciferase

as a reporter gene (cell lines 2209-23 and pRLucH771b75S/I,

respectively) were used as described by Le Pogam et al.²⁸

Transient HCV Replicon Assay Using a Panel of GT-1 NS5B

Clinical Isolates. The GT-1b Con1 and GT-1a H77 transient

replicons in which the wild type NS5B gene has been replaced by

NS5B isolates obtained from untreated patients were used as described

by Le Pogam et al.²²

HCV NS5B Polymerase Protein Crystallography. HCV NS5B

GT1b (BK 1-570) was expressed and puriﬁed as previously

described.²⁹Crystals of compound-bound polymerase were grown

by sitting drop vapor diﬀusion set using 7 mg/mL protein plus 5 mM

each respective compound over a reservoir containing 50 mM sodium

citrate (pH 4.9), 24% polyethylene glycol 4000, and 7.5% glycerol.

Crystals were transferred to a solution of mother liquor plus 20%

glycerol for cryoprotection and ﬂash frozen in liquid nitrogen. Data

were collected at the Advanced Light Source beamline 5.0.2 and

Stanford Synchrotron Radiation Laboratories beamline 7-1. Data were

reduced with HKL2000,³⁰and a molecular replacement solution was

determined with Phaser³¹and reﬁned using cycles of Buster^32,33with

manual rebuilding using Coot.³⁴

Hepatocyte Metabolic Stability Assay. The compounds were

incubated with approximately 1.0 × 10⁶cells/mL in suspension with

Williams’ medium E (supplemented with glutamine, antibiotics,

insulin, dexamethasone, and 10% FCS) at 37 °C in an atmosphere

of 5% CO₂. Hepatocyte incubations were done at diﬀerent

concentrations (0.1, 0.3, 1.0, 3.0, and 10.0 μM) of compounds on a

Teleshake 1 Variomag at speed of 900 rpm. Incubations were

terminated by the addition of 200 μL of methanol plus internal

To a solution of 1-(3,8-dibromo-5-methoxy-6-quinolyl)-

cyclopropanecarbonitrile (280 mg, 0.733 mmol) in CH₂Cl₂(14 mL)

was added DIBAL (0.87 mL, 0.87 mmol, 1 M in CH₂Cl₂) dropwise at

−78 °C. The reaction mixture was stirred at the same temperature for

1 h. The reaction was quenched with aqueous Rochelle salt and

extracted with CH₂Cl₂. The organic layer was washed with water, dried

over Na₂SO₄, ﬁltered, and concentrated. The crude material was

puriﬁed over SiO₂(gradient, 0−100% CH₂Cl₂in hexane) to aﬀord 1-

(3,8-dibromo-5-methoxy-6-quinolyl)cyclopropanecarbaldehyde (220

1

mg, 78%) as a white solid. H NMR (400 MHz, CDCl₃) δ 9.17 (s,

1 H), 9.01 (d, J = 2.2 Hz, 1 H), 8.60 (d, J = 2.1 Hz, 1 H), 7.89 (s, 1 H),

3.92 (s, 3 H), 1.77 (dd, J = 7.6, 4.9 Hz, 2 H), 1.56 (dd, J = 7.0, 4.4 Hz,

2 H).

To a solution of 1-(3,8-dibromo-5-methoxy-6-quinolyl)-

cyclopropanecarbaldehyde (630 mg, 1.64 mmol) in CH₂Cl₂(14

mL) was added diethylaminosulfur triﬂuoride (DAST) (1.05 g, 6.54

mmol), and the resulting solution was stirred at room temperature for

72 h. The reaction mixture was diluted with water and extracted with

CH₂Cl₂. The organic layer was washed with water, dried over Na₂SO₄,

ﬁltered, and concentrated. The crude product was puriﬁed over SiO₂

(gradient, 50−100% CH₂Cl₂in hexane) to aﬀord 3,8-dibromo-6-[1-

(diﬂuoromethyl)cyclopropyl]-5-methoxyquinoline (600 mg, 90%) as a

1

white solid. H NMR (400 MHz, CDCl₃) δ 9.0 (d, J = 2.0 Hz, 1 H),

8.57 (d, J = 2.1 Hz, 1 H), 8.07 (s, 1 H), 5.89 (t, J = 57.6 Hz, 1 H), 4.05

(s, 3 H), 1.35 (br t, J = 6.1 Hz, 2 H), 1.18−1.12 (br s, 2 H).

To

a microwave vial were added 3,8-dibromo-6-[1-

(diﬂuoromethyl)cyclopropyl]-5-methoxyquinoline (600 mg, 1.47

mmol), 4-(methanesulfonamido)phenylboronic acid (317 mg, 1.47

mmol), Pd(PPh₃)₄(162 mg, 0.14 mmol), Na₂CO₃(469 mg, 4.42

mmol), MeOH (9 mL), and toluene(3 mL). The vial was capped and

heated in the microwave reactor at 120 °C for 1 h. The reaction

mixture was ﬁltered, and the ﬁltrate was concentrated. The residue was

diluted with CH₂Cl₂, washed with water, dried over Na₂SO₄, and

concentrated. The crude material was puriﬁed over SiO₂(gradient,

30−80% EtOAc in hexanes) to obtain N-[4-[8-bromo-6-[1-

(diﬂuoromethyl)cyclopropyl]-5-methoxy-3-quinolyl]phenyl]-

1

methanesulfonamide (670 mg, 91%) as a solid. H NMR (300 MHz,

CDCl₃) δ 9.25 (d, J = 2.0 Hz, 1 H), 8.53 (d, J = 2.1 Hz, 1 H), 8.06 (s,

1 H), 7.73 (d, J = 8.4 Hz, 2 H), 7.41 (d, J = 8.5 Hz, 2 H), 6.69 (s, 1 H),

5.94 (t, J = 57.7 Hz, 1 H), 4.09 (s, 3 H), 3.11 (s, 3 H), 1.40−1.34 (br

m, 2 H), 1.21−1.13 (br m, 2 H).

To a ﬂask was added N-[4-[8-bromo-6-[1-(diﬂuoromethyl)-

cyclopropyl]-5-methoxy-3-quinolyl]phenyl]methanesulfonamide (520

mg, 1.05 mmol), tert-butyl carbamate (147 mg, 1.25 mmol),

Pd₂(dba)₃.CHCl₃(123 mg, 0.12 mmol), 2-di-tert-butylphosphino-

2′,4′,6′-triisopropylbiphenyl (172 mg, 0.4 mmol), sodium tert-butoxide

(301 mg, 3.14 mmol), and toluene (8 mL). The mixture was purged

with argon for 20 min and stirred at room temperature for 72 h. The

mixture was quenched with NH₄Cl and extracted with EtOAc. The

organic layer was washed with brine, dried over Na₂SO₄, ﬁltered, and

concentrated. The crude material was puriﬁed over SiO₂(gradient,

30−80% EtOAc in hexane) to give tert-butyl N-[6-[1-

(diﬂuoromethyl)cyclopropyl]-3-[4-(methanesulfonamido)phenyl]-5-

1

methoxy-8-quinolyl]carbamate (150 mg, 27%) as a solid. H NMR

(300 MHz, CDCl₃) δ 9.01 (d, J = 2.2 Hz, 1 H), 8.82 (br s, 1 H), 8.49

(d, J = 2.3 Hz, 1 H), 8.39 (br s, 1 H), 7.72 (d, J = 8.5 Hz, 2 H), 7.40

(d, J = 8.5 Hz, 2 H), 6.53 (s, 1 H), 6.13 (t, J = 58.4 Hz, 1 H), 4.02 (s, 3

H), 3.10 (s, 3 H), 1.56 (s, 9 H), 1.36 (dd, J = 6.2, 5.0 Hz, 2 H), 1.22−

1.14 (br m, 2 H).

To a solution of tert-butyl N-[6-[1-(diﬂuoromethyl)cyclopropyl]-3-

[4-(methanesulfonamido)phenyl]-5-methoxy-8-quinolyl]carbamate

(150 mg, 0.28 mmol) in CH₂Cl₂was added 4 M HCl in dioxane (1.55

mL, 6.2 mmol). The mixture was stirred at room temperature for 1 h

and then neutralized with saturated aqueous NaHCO₃and extracted

P

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Journal of Medicinal Chemistry

Article

standard after 2, 10, 30, 60, 120, and 180 min of incubation. Samples

were transferred to analytical plates and sealed. The supernatants were

analyzed by LC/MS/MS. All incubations were conducted in duplicate,

and the percentage of compounds remaining at the end of the

incubation was determined from the LC/MS/MS peak area ratio.

Pharmacokinetics. Male Wistar/Han (CRL:WI) rats (Charles

River Laboratories, Hollister, CA or Raleigh, NC) used in these studies

weighed between 180 and 300 g and were surgically implanted with

cannulas in the jugular and/or femoral veins. Male beagle dogs were

used in these studies. All studies were conducted under IACUC

approved protocols, and animals were allowed free access to food and

water. Single intravenous bolus doses were administered into the tail

vein or into the jugular cannula of dual-cannulated animals using a

dosing formulation consisting of 2% dimethylacetamide/20%

PEG400/23.4% HPBCD; the remaining cannulas were used for

blood collection. Single oral doses were administered by gavage using a

dosing formulation consisting of 2% Klucel LF in water. Blood was

collected into tubes containing EDTA at typically 0.08 (iv only), 0.25,

0.5, 1, 2, 4, 6, 8, and 24 h after dosing. Plasma concentrations were

determined by a liquid chromatography/tandem mass spectrometry

method (LC/MS/MS) following protein precipitation with acetoni-

trile.

Preparation of 41 Microprecipitated Bulk Product. Amor-

phous form of the compound was manufactured by solvent controlled

precipitation (SCP) method. The drug and Eudragit L100-55 polymer

(copolymers of acrylic and methacrylic acid) were dissolved in the

dimethylacetamide (DMA) by stirring at room temperature. The

solution was then added to the temperature controlled antisolvent

aqueous medium (at dilute HCl, pH 3.0, temperature 2−8 °C) that

allows rapid co-precipitation of drug and API. The residual DMA was

extracted with frequent washing, followed by ﬁltration and drying. The

formulation showed amorphous X-ray diﬀraction pattern. The yield

from SCP process was over 90%. Only Eudragit L100-55 polymer

provided stable amorphous formulation which stayed amorphous for

more than 60 days in the aqueous medium as well as under extreme

stress conditions of 40 °C/100% RH.

operated for the U.S. Department of Energy Oﬃce of Science

by Stanford University. The SSRL Structural Molecular Biology

Program is supported by the DOE Oﬃce of Biological and

Environmental Research and by the National Institutes of

Health, National Institute of General Medical Sciences

(including Grant P41GM103393), and the National Center

for Research Resources (Grant P41RR001209). The contents

of this publication are solely the responsibility of the authors

and do not necessarily represent the oﬃcial views of NIGMS,

NCRR, or NIH.

ABBREVIATIONS USED

■

DAA, direct-antiviral agent; HCV, hepatitis C virus; MBP,

microprecipitated bulk powder; NI, nucleoside inhibitor; NNI,

non-nucleoside inhibitor; NS5B, nonstructural 5B; GT,

genotype; SVR, sustained virological response

REFERENCES

■

(1) World Health Organization. Media Centre. Hepatitis C. http://

www.who.int/mediacentre/factsheets/fs164/en/ (accessed May 17,

2013).

(2) McGivern, D. R.; Lemon, S. M. Virus-Specific Mechanisms of

Carcinogenesis in Hepatitis C Virus Associated Liver Cancer. Oncogene

2011, 30, 1969−1983.

(3) European Association for the Study of the Liver.. EASL Clinical

Practice Guidelines: Management of Hepatitis C Virus Infection. J.

Hepatol. 2011, 55, 245−264.

(4) Asselah, T.; Marcellin, P. Direct Acting Antivirals for the

Treatment of Chronic Hepatitis C: One Pill a Day for Tomorrow.

Liver Int. 2012, 88−102.

(5) Tanwar, S.; Trembling, P. M.; Dusheiko, G. M. TMC435 for the

Treatment of Chronic Hepatitis C. Expert Opin. Invest. Drugs 2012, 21,

1193−1209.

(6) Gane, E. Future Prespectives: Towards Interferon-Free Regimens

for HCV. Antiviral Ther. 2012, 17, 1201−1210.

ASSOCIATED CONTENT

* Supporting Information

■

S

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Experimental procedures and spectroscopic data for com-

pounds 34, 40, 43−47, and 49 and their corresponding

synthetic intermediates; data collection and reﬁnement

statistics for cocrystals of NS5B with 41 and 48. This material

is available free of charge via the Internet at http://pubs.acs.org.

Accession Codes

Protein crystal structure coordinates and structure factor ﬁles

have been deposited in the Protein Data Bank under the

accession codes 4MIA (41) and 4MIB (48).

AUTHOR INFORMATION

Corresponding Author

*Phone: 650-279-9988. E-mail: franciscotalamas@gmail.com.

■

Notes

The authors declare no competing ﬁnancial interest.

(9) Sofia, M. J.; Chang, W.; Furman, P. A.; Mosley, R. T.; Ross, B. S.

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ACKNOWLEDGMENTS

■

We gratefully acknowledge all members of the Analytical

Department in Roche Palo Alto and the Physical Chemistry

Department at Hoﬀmann-La Roche, Nutley, NJ, for the

characterization of the compounds. The Advanced Light

Source is supported by the Director, Oﬃce of Science, Oﬃce

of Basic Energy Sciences, of the U.S. Department of Energy

under Contract DE-AC02-05CH11231. Portions of this

research were carried out at the Stanford Synchrotron

Radiation Lightsource, a Directorate of SLAC National

Accelerator Laboratory and an Oﬃce of Science User Facility

Q

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dx.doi.org/10.1021/jm401329s | J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of N-[4-[6-tert-butyl-5-methoxy-8-(6-methoxy-2-oxo-1 H -pyridin-3-yl)-3-quinolyl]phenyl]methanesulfonamide (RG7109), a potent inhibitor of the hepatitis C virus NS5B polymerase

DOI: 10.1021/jm401329s

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